Download Tearing down barriers: understanding the

Journal of Experimental Botany, Vol. 63, No. 17,
2, pp.
2012
pp.695–709,
6059–6067,
2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers288 Advance Access publication 4 November, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH
PAPER
Darwin Review
Tearing
downoceanica
barriers: cadmium
understanding
thechanges
molecular
In
Posidonia
induces
in DNA
mechanismsand
of interploidy
methylation
chromatin hybridizations
patterning
Nicole Schatlowski
and
Claudia Köhler*
Maria
Greco, Adriana
Chiappetta,
Leonardo Bruno and Maria Beatrice Bitonti*
Plant Biology
and Forest
Genetics,
UppsalaofBioCenter,
Swedish University
Agricultural
and LinneandiCenter
Department of Ecology,
University
of Calabria,
Laboratory
Plant Cyto-physiology,
PonteofPietro
Bucci, Sciences
I-87036 Arcavacata
Rende,of
Plant Biology,
Cosenza,
Italy 750 07 Uppsala, Sweden
* To whom correspondence should be addressed. E-mail: [email protected]
* To whom correspondence should be addressed. E-mail: [email protected]
Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011
Received 2 July 2012; Revised 20 September 2012; Accepted 24 September 2012
Abstract
Abstract
In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
Polyploidization, the process leading to more than two sets of chromosomes, is widely recognized as a major speciaepigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
tion mechanism that might hold the key to Darwin’s ‘abominable mystery’, as he referred to the sudden rise of angioits effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in
sperms to ecological dominance. On their way to become polyploid most plants take the route through the production
actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd,
of unreduced gametes that might eventually lead to viable triploid intermediates able to backcross or self-fertilize to
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach,
give rise to stable polyploid plants. Polyploids are almost instantly reproductively isolated from their non-polyploid
respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
ancestors; as hybridizations of species that differ in ploidy mostly lead to non-viable progeny. This immediate reprowas also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron
ductive barrier referred to as ‘triploid block’ is established in the endosperm, pointing towards an important but
microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de
greatly underestimated role of the endosperm in preventing interploidy hybridizations. Parent-of-origin specific gene
novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of
expression occurs predominantly in the endosperm and might cause the dosage-sensitivity of the endosperm. This
interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd
article illustrates, based on the recent molecular and genetic findings mainly gained in the model species Arabidopsis
perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are
thaliana, the ‘journey’ of unreduced gametes to triploid intermediates to polyploid plants and will also discuss the
linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin.
implications for interploidy and interspecies hybridizations.
Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
Key words: Epigenetics, genomic imprinting, hybridization barriers, parental conflict, polyploidy, speciation.
Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
Introduction
Introduction
Polyploidy, the presence of more than two sets of chromosomes (Ramsey and Schemske, 2002) than a natural advantage associ-
within a nucleus, is a widespread phenomenon among plant speIn the Mediterranean coastal ecosystem, the endemic
cies and recent nuclear genome sequencing projects suggest that
seagrass Posidonia oceanica (L.) Delile plays a relevant role
all angiosperms might have experienced polyploidization durby ensuring primary production, water oxygenation and
ing their evolution (Soltis et al., 2008; Jiao et al., 2011). This
provides niches for some animals, besides counteracting
observation in particular is striking since polyploidization has
coastal erosion through its widespread meadows (Ott, 1980;
been postulated an evolutionary ‘dead end’, with additional copPiazzi et al., 1999; Alcoverro et al., 2001). There is also
ies of genes masking deleterious as well as potential advantaconsiderable evidence that P. oceanica plants are able to
geous mutations and, therefore, escaping selection (Stebbins,
absorb and accumulate metals from sediments (Sanchiz
1971).
This hypothesis
has been
further
supported
the findet
al., 1990;
Pergent-Martini,
1998;
Maserti
et al.,by
2005)
thus
ing
that
recently
formed
polyploid
plants
diversify
at
lower
rates
influencing metal bioavailability in the marine ecosystem.
(Mayrose
et al., 2011)
show reduced
as evidenced
For
this reason,
this and
seagrass
is widelyfitness,
considered
to be
by
decreased
pollen
viability
and
seed
production
(Ramsey
and
a metal bioindicator species (Maserti et al., 1988; Pergent
Schemske,
2002).
The
high
frequency
of
polyploids
seems,
et al., 1995; Lafabrie et al., 2007). Cd is one of most
therefore, rather
a consequence
theirterrestrial
high rate and
of formation
widespread
heavy
metals in of
both
marine
environments.
ated with newly formed polyploids.
Although not essential for plant growth, in terrestrial
The establishment and maintenance of a new polyploid lineage
plants, Cd is readily absorbed by roots and translocated into
is challenging since various difficulties need to be dealt with,
aerial organs while, in acquatic plants, it is directly taken up
above all the disposition to problems in meiosis that can result
by leaves. In plants, Cd absorption induces complex changes
in unbalanced chromosome numbers (aneuploidy). Aneuploidy
at the genetic, biochemical and physiological levels which
can be fatal, although the degree of lethality varies among plant
ultimately account for its toxicity (Valle and Ulmer, 1972;
species ranging from complete lethality to a substantial tolerance
Sanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005;
(Henry et al., 2007). This contrasts with the situation in animals,
Weber et al., 2006; Liu et al., 2008). The most obvious
where most aneuploidies are embryo lethal (Matzke et al., 2003).
symptom of Cd toxicity is a reduction in plant growth due to
Nevertheless,
(even successful
aneuploidy)
can lead
an
inhibitionaneuploidy
of photosynthesis,
respiration,
and nitrogen
to
genomic
and
epigenetic
instability
(Papp
et al.,
1996),
two
metabolism, as well as a reduction in water and mineral
threats
that
polyploids
in
general
have
to
face
(Chen,
2010).
uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000;
Furthermore,
results
in a genome
increased in
Shukla
et al., polyploidization
2003; Sobkowiak
and Deckert,
2003).
size
that
needs
to
be
accommodated
in
the
nucleus
which
At the genetic level, in both animals and plants,might
Cd
can induce chromosomal aberrations, abnormalities in
© 2011
The Author
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
ª
The Author(s).
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email: distributed
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6060 | Schatlowski and Köhler
cause changes in the nucleus and in the overall cell architecture
(Comai, 2005). The existence of multiple copies of one gene can
lead to functional diversification, with one copy acquiring a new
function (neofunctionalization) or a partitioning of expression
patterns (subfunctionalization) that involves reciprocal (most
likely epigenetic) silencing of each of the copies leading to differential expression in different tissues (Lynch and Force, 2000;
Adams et al., 2003). Many polyploid species are also able to
reproduce asexually, either through somatic clones or apomixis,
therefore being independent of the availability of mating partners and timing issues. Apomixis, asexual reproduction through
seeds, comprises several aberrations to sexual reproduction
including embryo sac formation without meiosis, autonomous
embryo development, and adaptations in endosperm development and can be initiated at different stages in ovule development (Koltunow and Grossniklaus, 2003).
The potential of a newly formed polyploid species to establish itself successfully depends on the available opportunities to
colonize new habitats. Analysis of ancient polyploidy revealed
a clustering of genome duplications around the Cretaceous–
Tertiary extinction event approximately 65 million years ago
(Fawcett et al., 2009), supporting the view that environmental changes promote polyploid establishment (Oswald and
Nuismer, 2011).
This article will discuss mechanisms underlying the formation
of polyploid plants and their consequences for plant evolution.
We will start by reviewing the pathways leading to unreduced
gamete formation and then discuss the following hybridization
process and the mechanisms that establish interploidy hybridization barriers (the triploid block). We propose that the triploid
block acts as an instant reproductive barrier in the endosperm
that successfully prevents backcrossing of the newly established
polyploid plant with its progenitor. Therefore, the high rate of
speciation events in response to polyploidization is a consequence of the triploid block that might underlie the rapid radiation of flowering plants in the mid-Cretaceous.
Mechanisms of polyploid formation by
unreduced gametes
Polyploid plants can arise from a diploid ancestor through a
duplication or multiplication of a set of chromosomes within one
species (autopolyploidization) or through hybridization of different species followed by a genome doubling event (allopolyploidization). Whether auto- or allopolyploidy is more frequent among
flowering plants is still under debate (Mallet, 2007), although it
can be predicted that autopolyploidy is the more frequent form of
polyploidy because within-species mating is more frequent than
interspecific mating (Hegarty and Hiscock, 2008). However, a
strict classification proves to be complex, as the degree of divergence to the parents is highly variable and true ancestry can be
difficult to establish. Genome duplication events are estimated
to occur at relatively high frequency and can result from somatic
doubling of meristematic tissues as well as from zygotes,
through polyspermy, when multiple sperm cells fertilize an egg
cell. However, the most common mechanism on the way to polyploid formation is probably via unreduced gametes that contain
the full somatic chromosome set (2n) (Ramsey and Schemske,
1998). Unreduced gamete formation occurs with an estimated
frequency of ~0.5% per gamete, suggesting a high potential for
frequent polyploid formation (Ramsey and Schemske, 1998;
Brownfield and Köhler, 2011). The double fertilization process
in angiosperms requires the formation of haploid (1n) male and
female gametes through meiosis. During meiosis I the homologous chromosomes pair and recombine, thus ensuring a genetically diverse progeny, whereas in meiosis II the sister chromatids
are separated and, during cytokinesis, split into haploid meiocytes. Both processes are highly complex and abnormalities are
likely to result in non-viable gametes. Although a large number
of different plant species producing viable 2n gametes have been
identified and described (Bretagnolle and Thompson, 1995),
our understanding of the underlying molecular mechanisms has
only recently advanced with the discovery of meiotic mutants in
the dicot model plant Arabidopsis thaliana that form unreduced
gametes at high frequency. The genetic constitution of unreduced
gametes therefore depends on the meiotic stage at which the
defect occurs; mutants impaired in meiosis I produce gametes
containing two chromosomes of non-sister chromatids [and are
referred to as first division restitution (FDR) mutants]. Gametes
formed by a FDR mechanism are highly heterozygous, whereas
mutants faulty in meiosis II [second division restitution (SDR)
mutants] contain two sister chromatids and therefore show high
levels of homozygosity (Brownfield and Köhler, 2011). In the
following paragraph, examples are presented for A. thaliana
mutants affected at different stages of gametogenesis that produce viable gametes which can result in polyploid offspring.
During meiotic prophase I the homologous chromosomes
condense, pair, and recombine, orchestrated by several protein
complexes, to create bivalents (Osman et al., 2011). A disruption
of these processes often leads to the formation of univalents that
cannot be separated properly in later stages. The SWI1/DYAD
gene is required for sister chromatid cohesion and recombination (Mercier et al., 2001; Agashe et al., 2002) with different
swi1/dyad alleles showing different strengths in the effects on
female or male gametogenesis. Female meiosis in the dyad allele
is mitosis-like, resulting in a low frequency of viable unreduced
female FDR gametes that are able to be fertilized by haploid
male gametes, resulting in viable triploid seed formation (Siddiqi
et al., 2000; Mercier et al., 2001; Ravi et al., 2008).
After the completion of meiosis I the meiocytes have to enter
meiosis II without undergoing another round of DNA replication. This can only be achieved if the cell cycle progression is
tightly controlled by exact fine-tuning of the involved components, specifically cyclin-dependent kinases (CDK) and their
activity controlling cofactors, cyclins. Consequently, impaired
control of cell cycle progression by loss of A-type cyclin A1;2
(CYCA1;2) activity causes the formation of unreduced gametes.
Whereas weak, temperature-sensitive alleles of cycA1;2/tam
(tardy asynchronous meiosis) cause a delay in cell cycle progression of male meiocytes, null mutations affect male and, to a
lesser extent, female meiosis as well by skipping the second division, resulting in cells that still contain sister chromatids after
cytokinesis (Wang et al., 2004, 2010; d’Erfurth et al., 2010). The
failure to enter meiosis II and the resulting production of dyads
instead of tetrads can also be observed in meiocytes of the osd1
Interploidy hybridizations | 6061
mutant (omission of second division 1) with 100% penetrance
for male gametes and 85% for female gametes (d’Erfurth et al.,
2009) and, as for cycA1;2/tam mutants, unreduced (SDR) gametes of the osd1 mutants can produce viable triploid or, in case of
mating of unreduced male with unreduced female osd1 gametes,
tetraploid offspring.
Unreduced gametes can also arise through defective spindle orientation during meiosis II when the chromosomes are
regrouped prior to their physical separation in cytokinesis.
Two mutants with similar nuclear FDR restitution phenotypes
have been described so far: ps1 and jas1. Defects in AtPS1
(Arabidopsis thaliana Parallel Spindle 1) and JAS (JASON) lead
to abnormally oriented spindles in male meiosis II which are frequently fused or parallel aligned compared with the perpendicular orientation in the wild type (d’Erfurth et al., 2008; Erilova
et al., 2009; De Storme and Geelen, 2011). As a consequence of
spindle rearrangements chromosomes associated with different
spindles are in close physical proximity at the end of anaphase II
and are subsequently contained within one cell during cytokinesis. However, in both mutants, female meiosis is not affected as,
among the offspring, only diploid and triploid plants were found.
Another mechanism leading to the formation of unreduced
gametes involves inaccurate meiotic cytokinesis leading to the
formation of cells with multiple nuclei as, for example, in tes/
stud mutants (tetraspore/stud). TES/STUD encodes a kinesin
and its mutation leads to microtubule disorganization and a disruption of male meiotic cytokinesis. This results in spores that
contain four nuclei that can fuse, giving rise to pollen grains with
di-, tri or tetraploid nuclei that can produce offspring of different
ploidy level (Hülskamp et al., 1997; Spielman et al., 1997; Yang
et al., 2003).
A common feature of the mutants mentioned above is the
production of viable unreduced gametes and viable offspring
that differ in ploidy levels from their parents with being mainly
tri- or tetraploid, but other ploidy levels might also be observed
depending on the type of mutation. These mutants provide powerful tools to investigate the effects and the underlying mechanisms of the generation of polyploid plants.
Mechanisms of polyploid formation:
hybridization and triploid block
As discussed earlier, the formation of polyploid plants from
diploid ancestors can arise through auto- or allopolyploidization and mainly involves the generation of unreduced gametes.
Simultaneous generation of unreduced male and female gametes
(e.g. as can be found in the osd1 mutant; d’Erfurth et al., 2009)
could lead to autopolyploid plants and, although rather rare, the
spontaneous generation and hybridization of unreduced gametes
of different species could lead to allotetraploids (Bretagnolle
and Thompson, 1995; Ramsey and Schemske, 1998). By contrast with this one-step mechanism (also called bilateral sexual
polyploidization), a two-step mechanism (unilateral sexual polyploidization) that employs an intermediate step generating triploids (therefore called the triploid bridge hypothesis) and their
self-fertilization or back-crossing to diploids seems to be the
major route to polyploidization (Fig. 1; Ramsey and Schemske,
1998). Due to their unbalanced chromosome number triploids
are unstable and will face difficulties during meiosis and only
a small fraction of gametes will finally produce tetraploids
(Ramsey and Schemske, 1998).
To successfully hybridize—either between different species
of the same ploidy (homoploid hybridization) or between individuals of different ploidy levels (interploidy hybridizations)—
several obstacles need to be overcome, from prezygotic barriers
that prevent pollen from fertilizing ovules (e.g. by pollen incompatibility, a profound problem in interspecies hybridizations) to
post-zygotic barriers that lead to the arrest of seed development.
The formation of triploids requires overcoming the triploid block
that often leads to high rates of seed abortion in response to interploidy hybridizations (Marks, 1966; Köhler et al., 2010). For a
long time it had been suspected that the endosperm is responsible
for the triploid block (Brink and Cooper, 1947). Nevertheless,
only in recent years has the mechanistic basis underlying the
triploid block begun to be uncovered and the following paragraphs will summarize the insight gained by using the genetic
and molecular tools provided by the model species, A. thaliana.
Pollen grains of angiosperms contain one vegetative cell and
two sperm cells, one of which fertilizes the haploid egg cell to give
rise to the embryo and the second sperm cell fertilizes the central
cell which gives rise to the endosperm, the tissue that surrounds
and nourishes the embryo after fertilization (Costa et al., 2004).
In most angiosperms the central cell is homodiploid, resulting
in a triploid endosperm. This specific triploid constitution of the
endosperm, with two genomes contributed from the maternal
parent (2m) and one genome contributed from the paternal parent (1p) is crucial for proper seed development. Any alteration of
the endosperm 2m:1p ratio in interploidy crosses is likely to lead
to aberrations in endosperm development and therefore to seed
abortion, although this is not inevitable and depends on the genetic background (Johnston et al., 1980; Lin, 1984; Scott et al.,
1998; Dilkes et al., 2008). The observation that an increase in
maternal genome contribution leads to smaller seed size and larger seeds arise through paternal excess, is in agreement with the
predictions of the ‘parental conflict theory’ (Haig and Westoby,
1989) or ‘kinship theory’ (Trivers and Burt, 1999), that postulates
that increased maternal genome contributions reduces nutrient
flow to the embryo whereas increased paternal genome contributions promote nutrient flows to the embryo (Haig and Westoby,
1989). The differences in seed size that accompany unbalanced
parental contribution are a result of differences in endosperm
development (Fig. 2). Arabidopsis has a nuclear-type endosperm
common for most angiosperms in which, after fertilization, the
central cell undergoes multiple rounds of nuclear division, resulting in a syncytium, followed by a migration of nuclei to the periphery, around which radial microtubules begin to form (Costa
et al., 2004). The micropylar region that surrounds the embryo
starts to cellularize first, followed by cellularization in the central
endosperm region. Finally, cell walls form around nuclei of the
chalazal endosperm (Brown et al., 1999; Berger, 2003). The timing of endosperm cellularization impacts on final seed size and
while delayed endosperm cellularization causes enlarged seed
formation, early endosperm cellularization correlates with the
formation of small seeds (Scott et al., 1998). Interploidy hybridizations in Arabidopsis have opposing effects on the central
6062 | Schatlowski and Köhler
Fig. 1. Routes to polyploid formation. (A) Diploid plants give rise to haploid gametes (1n) resulting in diploid offspring (2n). (B) If
unreduced gametes (male or female) occur and unite with their haploid counterpart the progeny will be triploid. The gametes arising
from those plants can be either haploid (1n), diploid (2n) or aneuploid (1n+z) due to meiotic problems. During fertilization (by either selfing
or outcrossing), a number of different gamete combinations are possible including the formation of diploid offspring, therefore restoring
the initial wild-type situation. However, unreduced gametes can unite with gametes containing aneuploid chromosome sets (resulting in
aneuploid offspring) or with haploid gametes resulting in triploid offspring, both options might lead to viable progeny. If diploid male and
female gametes unite, viable tetraploid offspring arises demonstrating that triploid intermediates might serve as bridge to the formation
of tetraploids. (C) Unreduced male and female gametes can arise simultaneously giving rise to tetraploid offspring.
regulators of endosperm cellularization, the AGAMOUS LIKE
(AGL) transcription factor AGL62 and AGLs interacting with
AGL62. Whereas delayed endosperm cellularization in paternal
excess interploidy hybridizations correlates with increased levels
of AGL62 expression, the converse applies in maternal excess
interploidy hybridizations (Erilova et al., 2009; Lu et al., 2012).
Importantly, maternal loss of AGL62 function can partially suppress the triploid block by restoring endosperm cellularization
(Hehenberger et al., 2012), suggesting a central role of deregulated AGL62 expression in executing the triploid block response.
Expression levels of AGL62 and interacting AGL genes in
response to interploidy crosses have been anticorrelated with 24
nt siRNA (p4-­siRNAs) levels in the endosperm (Lu et al., 2012),
suggesting that changes in p4-siRNA levels might have a role
in the response to interploidy hybridizations. Whereas increased
levels of p4-siRNAs in response to maternal genome excess have
been correlated with reduced expression of selected AGL genes,
decreased levels of p4-siRNAs in response to paternal genome
excess have been correlated with increased AGL gene expression. Although attractive, it remains to be investigated whether
siRNA levels are indeed causally connected to AGL gene expression levels.
Apparently, the genotype of the maternal sporophytic tissue
can also modulate the triploid block by impacting on endosperm
cellularization. Mutants in the maternal effect transcription factor gene TRANSPARENT TESTA GLABRA2 (TTG2) suppress
triploid seed abortion by promoting endosperm cellularization,
suggesting a change in signalling molecule release or perception
due to the loss of TTG2 (Dilkes et al., 2008).
Together, the timing of endosperm cellularization is of crucial importance for viable seed formation. Changes in the timing
of endosperm cellularization in response to interploidy hybridizations will cause seed abortion, establishing the endosperm
as a tissue promoting species formation by acting as a dosage-­
sensitive hybridization barrier.
Deregulated imprinted genes probably
underpin the triploid block response
As outlined above, changes in the ratio of parental contributions
to the endosperm alter seed size depending on the direction of
parental genome excess, suggesting a parent-of-origin effect.
Genomic imprinting is an epigenetic phenomenon rendering
genes specifically expressed dependent on their parent-of-origin that probably provides the basis for parent-specific effects
on progeny development. Therefore, imprinted genes are likely
candidates being involved in establishing interploidy hybridization barriers. But, in addition to imprinted genes that have
a strong allele-preference in their expression, genes that only
have a biased expression of one of the parental alleles could also
have a central role in the response to interploidy hybridizations
Interploidy hybridizations | 6063
Fig. 2. Model depicting the impact of parental genome dosage on seed development. During the double fertilization of angiosperms,
female and male gametes (depicted in the left and middle columns, respectively) unite to give rise to seeds containing the embryo
which is surrounded by endosperm (right column). In diploid plants (middle panel) the female gametophyte contains the haploid (1n) egg
cell (EC) which is fertilized by one of the two haploid sperm cells (SC) of the male gametophyte to give rise to the diploid (2n) embryo.
The homodiploid (2n) central cell (CC) is fertilized by the second sperm cell, giving rise to a triploid (3n) endosperm with a contribution
of 2 maternal (m) and 1 paternal (p) genomes. This 2m:1p genome dosage might be required to allow the repression of target genes
by the FIS PRC2, e.g. AGL62, a major regulator of endosperm cellularization (Hehenberger et al., 2012). Unreduced female gametes
(upper panel) lead to the formation of triploid embryos, but the endosperm contains a genome contribution of 4m:1p. This results in
seeds much smaller than wild-type seeds, with early cellularizing endosperm that might be caused by reduced AGL62 expression (Lu
et al., 2012). The fertilization of female gametes with unreduced (2n) male gametes (lower panel) also results in triploid embryos (which
might be viable or not) and a 2m:2p contribution to the endosperm. These seeds are increased in size and show disturbed embryo
development and endosperm cellularization failure (only endosperm nuclei are visible, depicted as dots), probably as a consequence of
increased AGL62 expression (Erilova et al., 2009; Hehenberger et al., 2012). Schematic bars depicted in the gametes/seeds represent
sets of genomes, with the red bars referring to the female and the blue bars to the male origin. A, antipodal cells; S, synergids. The
red dotted lines in the graphs depicting AGL62 expression level symbolize the threshold level of AGL62 required to initiate endosperm
cellularization.
(Dilkes and Comai, 2004). Two of the very first imprinted
genes to be identified in plants were MEA (MEDEA) and FIS2
(FERTILIZATION INDEPENDENT SEED 2), with their expression being restricted to the maternal alleles (Kinoshita et al.,
1999; Luo et al., 1999; Vielle-Calzada et al., 1999). MEA and
FIS2 inhibit endosperm proliferation in the absence of fertilization and aberration in their activity leads to endosperm overproliferation and delayed cellularization, a phenotype similar to
triploid seeds resulting from paternal excess crosses (Chaudhury
et al., 1997; Grossniklaus et al., 1998; Kiyosue et al., 1999). The
phenotypic similarities correlate with similar transcriptional profiles of fis2 mutant seeds and seeds derived from paternal excess
interploidy hybridizations (Erilova et al., 2009; Tiwari et al.,
2010), suggesting a connection between deregulated function of
MEA and FIS2 and interploidy seed defects. Both proteins are
subunits of the evolutionary conserved FIS-PRC2 (Polycomb
Repressive Complex 2), a high molecular weight complex that
comprises histone methyltransferase activity by depositing a
trimethyl group on lysine 27 of histone H3 (H3K27me3). This
epigenetic mark can be recognized by downstream protein complexes which are implicated in the long-term maintenance of
repressed states of gene expression of their target genes. PRC2s
of different subcomponent composition have been found to be
necessary in different tissues and at different stages of the plant
6064 | Schatlowski and Köhler
life cycle, respectively, with the FIS PRC2 being endosperm-specific (Hennig and Derkacheva, 2009) resulting in a derepression
of PRC2 target genes in the endosperm (Erilova et al., 2009).
Expression of MEA and FIS2 is altered in response to paternal
excess interploidy hybridizations, however, expression changes
are accession-dependent, with MEA expression being increased
in Landsberg erecta (Ler) while decreased in Columbia (Col) and
the converse holds true for FIS2 expression (Erilova et al., 2009;
Jullien and Berger, 2010). Notably, both accessions strongly differ in their response to interploidy hybridizations, with the Col
accession reacting more severely to increased paternal genome
dosage compared with the Ler accession (Dilkes et al., 2008).
Therefore, whether regulatory differences in MEA and FIS2
expression are cause or consequence of accession-dependent
interploidy responses remains to be investigated. While there are
differences between diverse Arabidopsis accessions in response
to interploidy crosses, deregulation of FIS PRC2 target genes
seems to be a general phenomenon that is probably responsible
for the developmental abnormalities in the endosperm (Erilova
et al., 2009; Jullien and Berger, 2010, N Schatlowski, unpublished data). To decipher the underlying mechanisms causing
deregulation of FIS PRC2 target genes in response to interploidy
crosses remains a challenge of future investigations.
Imprinted genes are among those FIS PRC2 target genes
that are deregulated in response to paternal excess interploidy
hybridizations (Wolff et al., 2011), suggesting that deregulated
imprinted genes play a central role in the interploidy response.
This notion is supported by the fact that many imprinted paternally expressed genes encode chromatin modifying proteins,
which often act in multimeric complexes and are, therefore,
prone to be sensitive to misbalanced genome dosage (Birchler
et al., 2001).
Interploidy and interspecies post-zygotic
hybridization barriers that act in the
endosperm might involve the same
mechanism
Seed defects in response to diploid interspecies crosses are often
strikingly similar to defects observed in response to interploidy
crosses (Haig and Westoby, 1991). Interspecies hybridizations
can often be rendered compatible by increasing the ploidy of
one parent (Johnston et al., 1980), suggesting that interspecies
crossing barriers are built by quantitative rather than qualitative genetic differences. If this hypothesis is true, interspecies
and interploidy hybridization barriers that are established in the
endosperm should have a common mechanistic basis. This view
is supported by work revealing that, in response to hybridization of A. thaliana with the closely related diploid variety of
A. arenosa, similar sets of AGL transcription factors become
deregulated as in response to interploidy crosses in A. thaliana
(Erilova et al., 2009; Walia et al., 2009). By increasing the ploidy
of the maternal A. thaliana parent, viable seed formation can be
restored upon hybridizations with A. arenosa (Josefsson et al.,
2006), suggesting that a limiting factor contributed by the maternal A. thaliana parent is required to restore viable seed formation. Viable seed formation could also be promoted by maternal
loss of function of the central endosperm regulator AGL62 and
its interacting partners AGL90 and PHERES1 (Josefsson et al.,
2006; Walia et al., 2009). As mentioned before, loss of AGL62
can also suppress interploidy seed abortion in A. thaliana
(Hehenberger et al., 2012), pointing towards a common mechanistic basis of interploidy and interspecies hybridization barriers. Recent data have revealed a complex genetic network being
involved in controlling the interspecies hybridization barrier
between A. thaliana and A. arenosa (Burkart-Waco et al., 2012).
In this study, different Arabidopsis accessions were tested for
the ability to rescue hybrid seed abortion and revealed a large
set of minor-effect loci from the maternal Arabidopsis genome
controlling hybrid growth and viability. Whether the same loci
play a role in regulating the triploid block response remains to
be investigated.
Consequences of polyploidy for plant
evolution
Since the middle of the last century, polyploidy has been widely
recognized as an important mechanism of plant speciation
(Stebbins, 1950; Grant, 1981; Soltis and Soltis, 2000). It has been
generally assumed that polyploidy results in modified global patterns of gene expression and is a major source of developmental
novelty (Osborn et al., 2003; Adams, 2007). In agreement with
this view, there have been two whole genome duplication events
in ancestral lineages shortly before the diversification of extant
seed plants and angiosperms. As a consequence of ancestral
whole genome duplications regulatory genes important to seed
and flower development diversified and probably contributed to
the rise and eventual dominance of seed plants and angiosperms
(Jiao et al., 2011). This view, however, is contrasted by studies
revealing that polyploid speciation rates are significantly lower
than those of diploid species (Wood et al., 2009; Mayrose et al.,
2011). Instead, it has been estimated that about 15% of speciation
events are accompanied by a ploidy increase (Wood et al., 2009),
implying that the widespread occurrence of polyploid taxa is a
consequence of the high rate of polyploid formation, rather than
a result of subsequent increases in diversification rates of polyploid lines (Wood et al., 2009; Mayrose et al., 2011).
The sudden rise of angiosperms in the mid-Cretaceous was
referred to by Charles Darwin as ‘an abominable mystery’, as the
underlying mechanisms that might have caused the rapid radiation of angiosperms remained a mystery in his time. We propose
that the high rate of speciation events in response to polyploidization is a consequence of instantly erected reproductive barriers in
the endosperm in response to polyploidization that will successfully prevent backcrossing of the newly established polyploid
plant with its progenitor. These reproductive barriers are likely
to be built by misbalanced contributions of dosage-sensitive
regulators that will cause endosperm developmental failure and
seed abortion. Dosage sensitivity could arise as a consequence of
genomic imprinting, suggesting that a subset of imprinted genes
could act as speciation genes.
Nevertheless, the most frequent route to polyploid formation is
probably through unreduced gametes and individuals with irregular chromosome numbers (Bretagnolle and Thompson, 1995;
Interploidy hybridizations | 6065
Ramsey and Schemske, 1998), suggesting that post-zygotic
hybridization barriers in response to interploidy hybridizations
can be overcome, albeit at low frequency. Whether this survival
frequency is modulated by environmental factors remains to be
investigated.
Conclusion
Recent years have witnessed a dramatic increase in our understanding of how polyploidy has contributed to diversification
and speciation. New quantitative assessments of polyploid speciation and extinction rates provided the first quantitative verification of the ‘dead-end hypothesis’ proposed by Stebbins more
than 40 years ago (Stebbins, 1971). Accordingly, the high incidence of polyploid plants seems to be a consequence of diploids
speciating frequently via polyploidy rather than a general advantage of polyploids. Only particularly fit lineages of polyploids
may persist and become evolutionary successful. Thus, the most
prevailing question to be addressed in the near future concerns
the underlying mechanism(s) of polyploidy-mediated plant speciation. In particular, to unravel the genetic basis of interploidy
hybridization barriers underpinning polyploidy-mediated plant
speciation will foster our understanding of mechanisms driving
plant evolution. In addition, this knowledge will pave the ground
for designing genetic strategies to overcome interploidy hybridization barriers, holding immense potential for plant breeding.
Finally, genes establishing interploidy-hybridization barriers
might as well be responsible for establishing interspecies hybridization barriers; this hypothesis remains to be tested.
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