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Unique features of the plant life cycle and their consequences

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UNIQUE FEATURES OF THE

PLANT LIFE CYCLE AND THEIR
CONSEQUENCES
Virginia Walbot* and Matthew M. S. Evans‡
Continuous development, the absence of a germline, flexible and reversible cellular differentiation,
and the existence of haploid and diploid generations — both of which express genes — are
characteristics that distinguish plants from animals. Because these differences alter the impact of
mutations, animals and plants experience varied selection pressures. Despite different life-cycles,
both flowering plants and multicellular animals have evolved complex sensing mechanisms that act
after fertilization as ‘quality checks’ on reproduction, and that detect chromosome dosage and the
parent of origin for specific genes. Although flowering plant embryos escape such surveillance in vitro,
embryo success in the seed often depends on a healthy endosperm — a nutritive tissue that is
produced by a second fertilization event in which maternal and paternal gene contributions can be
monitored immediately after fertilization and throughout development.

DOUBLE FERTILIZATION
The process by which two cells
in a megagametophyte fuse with
two sperm (typically from the
same pollen grain) to produce
both a diploid embryo and an
accessory organ — the
endosperm. Double fertilization
is characteristic of angiosperms,
but also occurs in other taxa in
which the result is usually the
production of two embryos.
*Department of Biological
Sciences, Stanford
University, Stanford,
California 94305-5020, USA.
‡
Department of Plant
Biology, Carnegie Institution
of Washington, 260 Panama
Street, Stanford, California
94305, USA.
e-mail:
walbot@stanford.edu;
mmevans@andrew2.
stanford.edu
doi:10.1038/nrg1064
The complete genome sequences of complex animals
and plants indicate that a similar number of genes
is required for a fruitfly, human and rice plant1.
Furthermore, many gene classes are conserved although
it is >1 billion years since the last shared progenitor of
plants and animals1. There are, however, fundamental
differences in the life cycles of plants and animals (FIG. 1)
that alter both the timing and the stringency of natural
selection in these kingdoms. The presence of genetically
active haploid (gametophyte) and diploid (sporophyte)
phases of the life cycle, the absence of a germline, and
a DOUBLE FERTILIZATION event during sexual reproduction
to produce both an embryo and a nutritive tissue
(ENDOSPERM) are unique features of flowering plants.
These plant-specific life-history components alter the
impact of mutations and present opportunities for dis-
tinctive developmental regulation. Here, we illustrate
these features of plant development using examples pri-
marily from Arabidopsis and maize that we compare
with animals, and comment on the resulting genetic
and evolutionary consequences of the plant life cycle.
In plants, the gametophytic stage presents an oppor-
tunity for natural selection on the haploid genome — as
in other haploid organisms, recessive deleterious alleles
that would be masked in diploid organisms are removed
from the population2. Over evolutionary history, there
has been a steady reduction in the diversity of structures
during the haploid phase; for example, gametophytes in
flowering plants are non-photosynthetic and contain few
cell types. Nonetheless, on the basis of RNA hybridization
kinetics, 20,000–25,000 genes are expressed in the pollen
of maize and Tradescantia3,4; this represents roughly one-
third to one-half of the probable total gene number5. We
propose that supplementary mechanisms have also
evolved to bolster the ‘quality check’ of the genome.
These include imprinting — the silencing of either
paternally or maternally derived alleles — as well as
checks on the ratio of maternal to paternal endosperm
ploidy and chromosome-arm dosage. In maize and
Arabidopsis, these quality checks ensure that sexual
reproduction is tightly regulated, in contrast to the innate
flexibility of plant development. These quality-checking
mechanisms could be seen to parallel the sensitivity of
mammalian development to perturbations in chromo-
some dosage, and the requirement for both a sperm and
an egg. Also, imprinting extends the haploid sufficiency

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consequence, plants probably have a lower genetic load

a b
than animals, the gamete properties of which are deter-
mined almost entirely by gene expression in progenitor
Zebrafish Tassel diploid cells. Recessive lethal and deleterious alleles can
Ear
accumulate in diploid animal populations, because they
Zebrafish
are masked in the heterozygous diploid individuals10.
Despite its possible advantages, natural selection has
favoured the reduction of the haploid phase in plants in
Mature sporophyte (2n) three ways: the proportion of the total life cycle, physical
Meiosis
size and the range of biological processes. Although
Egg (n) Sperm (n) Megaspore (n) Microspore (n) there is great diversity in algal life cycles11, in the green
Mitosis Mitosis alga Chlamydomonas reinhardtii, which is a model plant
for genetic analysis now that the nuclear genome is
Megagametophyte (n) Microgametophyte (n) sequenced12, most of the life cycle is haploid — gametes
(germinated pollen grain)
Egg cell fuse to produce a diploid cell, which can immediately
undergo meiosis to reconstitute haploid cells that prolif-
Polar Vegatative
nuclei erate by mitosis. The haploid phase expresses most of
nucleus
Sperm nuclei
the genome, whereas the diploid phase is centred on
Fertilization meiosis13. In the mosses and liverworts, which are basal
green plants, the haploid phase is also dominant.
Endosperm (3n) Individual gametophytes are free-living, AUTOTROPHIC and
sustain the diploid sporophytes, which remain embed-
Primordial ded in the gametophytes11. In flowering plants, this state
germ cells Embryo (2n)
is completely reversed as the haploid phase usually con-
sists of 2–7 cells that depend on the diploid parent for
nutrient acquisition11. Therefore, given the reduction in
Figure 1 | Comparison of plant and animal life cycles. a | Animal life cycle, in which gametes size and metabolic contribution in the most recently
are produced directly through meiosis, and gene expression is restricted to diploid cells. In the
evolved plant taxa, has the scope or intensity of the
embryo, the germline is set aside early (primordial germ cells) and these are the only cells that are
competent to undergo meiosis in the adult. b | Typical flowering plant life cycle (maize), in which
haploid sufficiency test diminished?
meiosis, followed by mitotic divisions, produces two types of haploid organism that are Despite the limited size and lifespan of pollen,
genetically active — the female megagametophyte and the male microgametophyte (pollen RNA transcripts that correspond to many genes are
grain). Mitotic division in the microgametophyte results in a pair of sister cells that differentiate into present, and, in some cases, there is proof that tran-
sperm. In the megagametophyte, a haploid egg differentiates and is fertilized by one sperm cell to scription occurred post-meiotically from the haploid
produce an embryo, and two haploid nuclei fuse to form a diploid central cell that is fertilized by genome3,4. Many genes are required to produce func-
another sperm cell to become triploid endosperm. The endosperm is a terminal nutritive tissue
tional pollen, as is clear from random mutagenesis
that contributes to normal embryo development in flowering plants (note that double fertilization
has been documented in some non-flowering plants86, and endosperm ploidy in flowering plants experiments. In Arabidopsis, ~1.3% of mutated lines
can be diploid or other ploidy values that are distinct from triploid87). Red arrows show plant- are gametophytic lethal, which indicates that at least
specific features. Photograph provided by E. Raz; figure reproduced with permission from REF. 88 200 genes are vital for gametophyte development14.
 (2002) Company of Biologists Ltd. This is probably an underestimate, as seed is not recov-
ered if either gametophyte fails before competent
gamete formation in the self-fertilizing flowers of
test transiently in the early stages of the diploid phase of Arabidopsis 15,16. In maize, the microgametophytes and
the life cycle. Similarly, in mammalian embryos, imprint- megagametophytes develop in separate INFLORESCENCES
ing creates effective haploid selection, but only for a — the tassel and ear, respectively17. After Mu transpo-
ENDOSPERM
small number of loci early in embryo development6. son mutagenesis of maize, complete male sterility
A tissue, found in flowering
plants, which is generated by the (100% defective pollen, which indicates a sporophytic
fusion of the central cell of the Selection on haploid cells failure) and semi-steriles (50% pollen death presum-
megagametophyte and a sperm. Plants have two phases to their life cycle: the diploid ably as a result of the segregation of defective genes
In most angiosperms, the sporophytic stage that ends in meiosis to produce hap- required in the microgametophyte) are among the
endosperm is triploid, with two
genome equivalents from the
loid cells, and the haploid gametophytic phase in which most common phenotypic classes18.
maternal line and one from mitotic proliferation produces a haploid plant that There is also overlap in the genes that contribute to
the paternal line; however, there includes the differentiation of a subset of cells as gametes. gametophytic and saprophytic success. For a few meta-
are many exceptions to this Both the egg-producing haploid plant (the megagame- bolic characteristics, including alcohol dehydrogenase
general rule.
tophyte) and the sperm-producing haploid plant (the activity and starch composition3,18, there is clear evi-
AUTOTROPHIC
microgametophyte) are genetically active —therefore, dence of selection that occurs on the gametophyte for
Able to independently acquire the phenotype reflects the haploid genotype. sporophytic traits. This could be widespread for many
a nutrient. It is often argued that haploid individuals are at a developmental regulatory genes.
selective disadvantage compared to diploid individuals Patterson and others exploited >850 reciprocal
INFLORESCENCE
The reproductive portion of a
as deleterious recessive mutations are exposed7,8, but translocation stocks, representing 1,700 deficiencies
plant that bears a cluster of an advantage is that haploidy is an opportunity to in maize, and established that they all caused pollen
flowers in a specific pattern. purge the population of deleterious mutations9,10. As a abortion and that ~90% resulted in megagametophyte

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Box 1 | Identification of loci essential for gametophyte development
The identification of loci that are required for the gametophyte phase and
gametogenesis requires special tools, because natural and induced mutants that are
gametophytic lethal are lost in standard mutagenesis approaches, even if they are viable
in heterozygotes. As gametophytes inherit either the functional or lethal allele, a semi-
sterility phenotype is observed, in which 50% of pollen or eggs are viable. If knockouts
result in gametopytic lethality, no diploid organism is generated and, therefore, it is
impossible to score the effects of mutations in diploid tissues.
Similarly, genes that cause lethality early in embryo development must be rescued to
uncover their functions at a later stage in life. To do this, transgenes that are expressed
selectively at the earlier stage (haploid or embryo) allow development and, therefore,
allow the evaluation of phenotypes at later stages. A recent example of this strategy
showed that the FERTILIZIATION INDEPENDENT ENDOSPERM (FIE) locus of
Arabidopsis is responsible not only for suppressing precocious endosperm development
but also for suppressing inappropriately early flowering81.
lethality19. Presumably, some mutations that cause nutri-
tional defects that are lethal to pollen, which is sealed
from metabolic exchange with the surrounding diploid
tissues for days, can be compensated for in megagameto-
phytes, which continue to absorb nutrients from the veg-
etative plant. In BOX 1, we describe how genes that are
involved in gametophyte development can be studied.
Gametophytic lethality might be considered surpris-
ing, given the extent of gene duplication in flowering
plant genomes. Autotetraploidy (chromosome doubling
in a species) and allotetraploidy (chromosome doubling
after a cross between two species) are common events in
all taxa. Approximately 70% of the Arabidopsis genome
consists of an ancient (>100 million years (Myr) ago)
duplication20,21. Furthermore, ~17% of Arabidopsis loci
have a linked recent duplication22. In maize, the modern
genome is the result of a complete duplication in the past
11–16 Myr23. At the time of genome-wide duplications,
gametophytic functions must be redundantly repre-
sented by a minimum of two loci, yet today maize is
effectively a diploid species for many loci because muta-
tions at one locus in a gene family produce a visible phe-
notype18. Such observations are, at present, discussed in
the context of the subfunctionalization model24, in
which the main mode of divergence of duplicate genes
involves the loss of portions of the gene-expression pro-
gramme at individual loci (or more precisely, their con-
stituent alleles). As a result, both loci are required during
the life cycle, because at particular stages only one locus
is expressed. For animals, subfunctionalization is
invoked to explain selection that favours the retention of
duplicate genes that act in different cell types; in plants,
SOMA
subfunctionalization could also have acted to distinguish
The cells of the body that cannot loci that are essential for the haploid phase from those
undergo meiosis. In plants, this that are now expressed in the diploid phase. Testing this
comprises the entire plant body idea requires locus-specific methods to distinguish
until the late specification of
between the expression patterns of closely related genes.
reproductive cells in flowers. By
contrast, animals have a somatic
body and a germline that Plants produce meiotic cells late in development
differentiates early in A crucial distinction between plants and animals con-
development; at reproductive cerns the germline. In animals, germ cells are desig-
maturity, the germ cells
proliferate, undergo meiosis and
nated early in embryo development and remain as a
the meiotic products separate stem-cell population throughout the life of the
differentiate into gametes. animal25; the germ-cell derivatives are the only cells that
NATURE REVIEWS | GENETICS
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REVIEWS
can undergo meiosis to produce eggs and sperm. Germ-
cell specification occurs by several mechanisms, includ-
ing the differential distribution of material stored in the
egg (in worms and flies) and fate decisions at the time
of gastrulation (in mammals)26,27. By contrast, the stem-
cell population that directs the growth of the plant
shoot only produces somatic structures — leaves and
shoots — until environmental and developmental sig-
nals trigger the switch to reproduction28,29; that is, the
apical stem cells switch from producing strictly somatic
organs to producing flowers, in which a few cells are
programmed to undergo meiosis to produce mega-
gametophytes, but many more cells are programmed to
differentiate into microgametophytes11. There is high
regularity in the anatomical placement of pre-meiotic
cells in most flowers, but this does not reflect a strin-
gent cell-lineage relationship — plant development is
generally governed by positional information (for
example, cell–cell interactions) that results in a high
regularity of structure28.
In plants, we are beginning to define the genes that
are crucial for the cell-fate decision that, at a particular
mitosis, restricts one cell to the SOMA and the sister cell to
the pre-meiotic fate. Among the handful of defined
genes that alter cell-fate determination in the anthers
of Arabidopsis are two leucine-rich repeat receptor
kinases30,31 that, if mutated, result in an excess of meiotic
cells and a deficiency of somatic cells. A nuclear protein
of unknown role is required for meiotic-cell specifi-
cation32. Several other loci that alter anther cell fate have
been defined in Arabidopsis but are not yet cloned33, and,
in maize, the multiple archesporial cells 1 (mac1) muta-
tion disrupts the somatic-to-germinal switch in both
male and female flowers34, which results in an excess of
meiotic cells. Also, genes that are expressed in the
megagametophyte are important during post-meiotic
and post-fertilization development35 — a theme that we
return to in the discussion of imprinting.
The failure to set aside a germline provides a poten-
tial problem for plants, because gametes could carry
many mutations that have accumulated during somatic
growth. This would create an incipient disaster for ani-
mals, given the absence of haploid selection. Somatic
diversification occurs in plants, as is readily observed in
mangroves in which the developing seeds germinate
while they are attached to the maternal plant; this
allows observation of the segregation of albino mutants
on some branches but not others36. Similarly, plants
that carry a transposable element inserted in an antho-
cyanin pigment gene can have red sectors that represent
somatic reversion; if red sectors occur in flowers, a
revertant red allele can be transmitted to the next gen-
eration37. Stringent selection during the haploid
gametophytic phase and selection during somatic
growth — as cell lineages carrying deleterious muta-
tions are impaired in growth and development and so
are less likely to contribute to gamete formation —
might abrogate the problem of accumulated mutations
before meiosis38. As reviewed in BOX 2, it has been
argued that these features make plants more tolerant of
somatically active transposable elements39.
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APOMIXIS Plasticity of plant embryogenesis gametophyte fate — indeterminate gametophyte (ig).

The production of seed without
embryo fertilization, which can
involve direct embryogenesis
from somatic cells or the
development of meiotic
products into embryos.
GYNOGENETIC
An individual that develops
from a cell in the
megagametophyte (typically the
egg) and, therefore, contains
only maternal chromosomes.
ANDROGENETIC
An individual that develops
from a sperm and, therefore,
contains only paternal
chromosomes.
Formation of a mammalian embryo requires either
the fertilization of an egg by a sperm or, nowadays, the
implantation of a somatic nucleus in the cytoplasm of
an egg40. In hundreds of insect41 and a few vertebrate42
species, sexual reproduction is circumvented by the
direct development of embryos from eggs. In plants,
reproduction is innately more flexible. There are many
examples of asexual plant reproduction, ranging from
the production of miniature plantlets on leaf margins,
as seen in Kalanchoë (FIG. 2), to APOMIXIS in which seeds
can develop in flowers without egg fertilization.
Apomixis occurs in diverse taxa43, and seeds can be pro-
duced if either somatic cells or meiotic products
undergo embryogenesis43,44. Further developmental
plasticity is observed in vitro, as diploid somatic cells45
and haploid pollen grains46 can form embryos in tissue
culture. Although haploid plants are sterile, chromo-
some doubling can produce fertile homozygous plants.
Therefore, a haploid genome of paternal origin is fully
competent to organize an embryo in tissue culture, and
an endosperm is dispensable for development outside
the context of a seed.
The spontaneous production of haploid embryos
in situ is line dependent in maize47,48, but the frequency
is generally 10–3 for GYNOGENETIC origin and 10–5 for
49
ANDROGENETIC origin . Therefore, neither the maternal
nor the paternal genome is absolutely required for
maize embryo development in a flower. Further
insights into the in semine (in seed) requirements
come from the analysis of an important regulator of
This maize gene seems to suppress the capacity of hap-
loid cells to form an embryo and to help maintain the
proper ploidy of megagametophyte cells. In loss-of-
function ig mutants, a percentage of the progeny are
haploid, and androgenetic haploids are as frequent as
the gynogenetic derivatives49. Curiously, in all cases,
the ig haploid plants have a normal triploid endo-
sperm, which indicates that endosperm fertilization
has occurred. Therefore, in maize, a haploid embryo
probably needs a normal triploid endosperm to sur-
vive. Interestingly, even in plants that produce seed by
apomixis, most taxa produce an endosperm by fertil-
ization of the central cell by a sperm cell50. However,
endosperm is probably not required in all flowering
plants, with an extreme case being orchids, in which a
second fertilization usually occurs but the triploid
endosperm nucleus quickly degenerates51.
Non-equivalence of parental genomes
In most angiosperms, both an embryo and its endo-
sperm are part of a developing seed — the unit of repro-
ductive dispersal (FIG. 3). The functional relationship of
the endosperm to the embryo parallels that of the pla-
centa to the fetus in mammals, because the endosperm
nourishes the embryo. Several lines of evidence show
that seed production requires contributions from both
parents, which indicates that the maternal and paternal
genomes are not equivalent. Parent-of-origin effects can
be detected by varying the relative germplasm contribu-
tions from the two parents and by examining mutations

Box 2 | The plant life cycle establishes conditions for tolerating transposons
The combination of haploid selection and the production of gametes from diverse somatic lineages might allow plants
to harbour active transposons with few consequences. Obviously, human selection for transposon activity at colour
genes resulted in the diversity of striped and dotted maize kernels, and of flecked horticultural flower varieties. But
transposon excision from non-vital colour genes should be accompanied by insertions elsewhere. Insertions in essential
genes should result either in selection against transposon activity or the loss of viability in the line, because many new
insertions disrupt gene function. Newly generated insertion alleles contribute to the somatic mutations in the plant, but
can only accumulate in the population if they survive haploid selection. The combination of purifying selection of the
haploid phase and the diversity of somatic lineages that can generate flowers, buffers the plant from transposon excess.
New dominant-lethal mutations in presumptive germ cells do not eliminate reproduction elsewhere, and lethality in the
gametes removes deleterious alleles from the population.
The more permissive conditions for ‘genome experimentation’ in plants might help explain the enormous range of
genome sizes, even among closely related taxa. Genome composition analysis shows that, in many cases, genome size
increases reflect the recent amplification of transposable elements82,83.
In some cases, transposon behaviour is directly tied to important features of the plant life cycle. Mutator activity in
maize results from the mobility of MuDR/Mu elements, which collectively can increase forward mutation frequency
50–100-fold84. These highly active multicopy transposons selectively insert in maize genes, but do not kill the host. The
MuDR/Mu transposon family is mobile only late in the life cycle during the last few somatic cell divisions; this
minimizes the impact on somatic growth, because only a few cells will carry any newly arising insertion mutant.
Furthermore, each transposon excision and insertion generates two chromosome breaks, but this genotoxic stress does
not impact the stem-cell populations or organs at early stages of their development.
By contrast, the Ac/Ds and Spm element families studied by Barbara McClintock are active at all somatic stages, but as
they are present in only one or a few copies, their activities cause only a modest disruption compared with what might
happen if 50–100 Mu elements were mobile throughout the life cycle84. Because of the late onset of all MuDR/Mu
activities, heritable Mu insertions occur in the pre-germinal cells before meiosis as well as in gametophytes, with 20% of
the events occurring after the last mitotic division that separates the two sperm85. Consequently, Mu transposons
amplify their copy number and create many new insertional mutants coincident with the stringent check of genome
quality that occurs in the haploid phase.

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Figure 2 | Seedlings produced along the Kalanchoë leaf margin. This is an example of the
flexibility in plant reproduction, as plantlets form along the leaf margin. Reproduced with permission
from Curtis Clark © (2003) Curtis Clark, Biological Sciences, California State Polytechnic University,
Pomona, USA.
that cause aberrant development if inherited through
one parent but not the other. There are several possible
mechanisms for parent-specific mutant phenotypes;
these include imprinting, a requirement for a particular
dose of active gene copies in the endosperm and the
cytoplasmic inheritance of gene products from one of
the haploid gametophytes.
a Maize seed b Arabidopsis seed
Endosperm
Embryo
Embryo
1 mm 60 µm
Figure 3 | Relative contribution of embryo and endosperm to mature seeds. a | A median
longitudinal section of a mature maize seed, stained with Evans blue to show the programmed
cell death of the starchy endosperm. The bulk of the seed consists of the endosperm rather than
the embryo. b | By contrast, in the median longitudinal section of a mature Arabidopsis seed, the
embryo almost completely fills the seed coat and little endosperm persists. Reproduced with
permission from J. A. Long and M. K. Barton, Carnegie Institution, Stanford, California, USA.
NATURE REVIEWS | GENETICS
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Although all flowering plants must produce a suc-
cessful embryo, species differ substantially in the relative
importance of the endosperm (FIG. 3); they range from
having no endosperm, as in orchids52, to species with a
transitory organ that supports embryo development,
and to cases in which the endosperm stores the nutri-
tional reserves for seedling establishment. In Arabidopsis,
the endosperm is crushed by embryo development and
its nutrients are transferred to the embryo. In cereals,
such as maize, the endosperm can be 90% of the seed
weight, extending the reliance of the embryo on the
endosperm to seedling germination. Given the variabil-
ity of the reliance of the embryo on the endosperm, we
might expect variability in the extent of parent-of-origin
effects on seed development.
What is the function of parent-of-origin effects? We
have already introduced the concept that the extension
of functional haploidy after fertilization could serve as a
further check on genome quality. Consistent with this,
evidence now points to functional haploidy for maternal
alleles acting early in endosperm and embryo develop-
ment, and this could effectively extend haploid selec-
tion53. Also, parent-of-origin effects act as a quality
check on sexual reproduction, because seed is only pro-
duced if the fertilization of appropriately programmed
male and female gametes occurs.
An additional explanation for parent-of-origin
effects originates from the parental conflict theory, in
which maternal and paternal alleles are differentially
expressed after fertilization to modulate resource alloca-
tion to the progeny52. The main prediction is that there
should be specific loci in which either the maternal or
the paternal allele is expressed after fertilization. The dif-
ferences in parental contribution to seed success are
substantial: the paternal parent provides little beyond
chromosomes, whereas the maternal parent supplies the
nutrients for embryo and endosperm development, and
maternal tissues form the seed coat and the surrounding
fruit tissues. The parental conflict theory proposes that
paternal alleles evolve to maximize nutrient allocation
to the individual seed (the selfish paternal parent),
whereas the maternal parent must support a suite of
seeds that develop in a fruit; so, maternal alleles are
probably under selection to reduce resource allocation
and avoid a negative impact on the overall reproductive
potential of the mother. The genetic basis for the con-
flict between the parents is that the maternal plant is
equally related to each embryo and endosperm, and,
therefore, has a similar investment in each seed. By con-
trast, if there are different paternal parents, individual
seeds are not full siblings and so are in competition for
maternal resources52.
The substantial evidence that specific chromosome
arms of paternal origin are essential for maize endo-
sperm development — which implies either that the
dosage of some genes is crucial, or that paternal alleles
are expressed whereas the corresponding maternal alle-
les are less effective — fits with the parental conflict
theory. Paternal deficiency for 8 of the 19 maize chromo-
some arms that have been tested using B-A chromosome
translocations (FIG. 4), resulted in a severe reduction in
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Mitosis I Mitosis II

A B
A B B A
B A B A

Generative cell Sperm

A B cells
A B
B A
Vegetative nucleus

Microspore
nucleus
A B A B
B A B A

Figure 4 | Microgametophyte development and B-A chromosome behaviour. The B chromosomes in maize are peculiar; they
carry no essential genes but can undergo translocation with the standard maize A chromosomes. In this example, the meiotic product
inherited one copy of the A-B and B-A chromosomes that had participated in an exact reciprocal exchange at the centromere. The
first mitotic division (mitosis I), produces a vegetative cell and a smaller generative cell, which have the same genetic constitution.
The generative cell then divides (mitosis II) to produce two sperm, each of which participate in fertilization. B-A chromosome non-
disjunction during this mitotic division routinely produces one sperm with an A-arm deficiency, whereas the second sperm has a
duplication for this arm. This enables the ploidy of A-chromosome arms to be manipulated. The vegetative cell — which supplies
most or all of the metabolic functions — has a balanced chromosome set, and, therefore, gametophyte viability is generally unaffected
by these translocations. B-A chromosome translocations in maize have been used to investigate the effect of paternal deficiency of
different chromosome arms54,55, and to mark different sperm nuclei to assess their fate65.

DICOT
A flowering plant with two
embryonic initial leaves, known
as cotyledons.
MONOCOT
A flowering plant with a single
cotyledon in the embryo.
endosperm size54,55. Increasing the number of maternal
copies did not overcome these defects and could exacer-
bate them56. Collectively, the data from Arabidopsis and
maize support the parental conflict model, in which
paternal alleles control the growth of the endosperm
because their expression is required or is dominant to
the maternal alleles.
The function of stringent ploidy ratio requirements
for endosperm formation could be linked to the parental
conflict theory, and could also provide a quality check to
ensure that viable seed is formed. FIGURE 1 shows that
flowering plant gametophytes have two cells that can
fuse with a partner of the opposite sex, resulting in a
diploid embryo and a typically triploid endosperm.
One hypothesis, which results from the parental conflict
theory, is that if the ploidy of the endosperm is altered,
the competition between the maternal and paternal
genomes is disrupted, which results in aberrant devel-
opment. These predictions have been tested using ig1
maize mutants, which produce megagametophyte cen-
tral cells with normal diploidy, or in many cases higher
ploidy, which shows that the endosperm that is pro-
duced after fertilization has a range of ploidy values.
Endosperm with a ratio of maternal to paternal genomes
of 2:1 (2m:1p) are normal, whereas 3m:1p endosperms
are viable but abnormal. Higher maternal ploidy
(tetraploid to octoploid) is lethal to the endosperm and
results in maize kernel abortion57. However, it is the
ratio of maternal:paternal genomes that is crucial for
endosperm development, not ploidy per se, as both
4m:2p and 2m:1p ratios promote normal endosperm
development57.
Similar results are seen in interploidy crosses in other
DICOT and MONOCOT plants, including wheat, barley and
species of Solanum and Primula 50,58. In Arabidopsis,
there is a greater tolerance for deviations in the maternal
to paternal genome ploidy ratio59, but normal develop-
ment only occurs in crosses producing endosperm
with a 2m:1p ratio. Furthermore, the phenotype of the
endosperm in these interploidy crosses fits the predic-
tions of the parental conflict theory — in which the rel-
ative contribution of the maternal and paternal parent
must be balanced to allow normal development — for
many of the species tested50,58. In these interploidy
crosses, both the endosperm and embryo ploidy ratios
are altered and, therefore, these experiments do not
resolve whether the endosperm or the embryo is the
crucial tissue for sensing parental contributions,
although the phenotype is seen in the endosperm. Only
in maize has it been shown that the endosperm ploidy
ratios, separate from those of the embryo, are crucial for
normal development57.
Parent-of-origin effects of individual loci
Finer resolution of the regulation of parent-of-origin
effects is possible if the focus switches to individual genes.
Imprinting breaks one of Mendel’s laws — namely that
alleles are unchanged by their mode of transmission. For
imprinted alleles, the parent of origin determines expres-
sion in the next generation. The first documented case of
gene imprinting 60 was the maize R regulatory gene,
which encodes a basic helix–loop–helix transcription
factor and controls the pigmentation of the endosperm
epidermis61. Maternal transmission of R in a cross with
an r male, results in an RR maternal/r paternal endo-
sperm with a solid red epidermis. In the reciprocal cross
involving an r female, however, the rr/R endosperm is
white with red sectors. A requirement for two doses of R

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POLYCOMB
A class of proteins — originally
described in Drosophila
melanogaster — that repress the
expression of the genes with
which they are associated. There
are several classes of polycomb
proteins and in higher plants
they are organized into gene
families.
NATURE REVIEWS | GENETICS
for full pigmentation can be ruled out using B-A translo-
cations (FIG. 4) to boost R copy number in the sperm —
in the rr/RR genotype the kernels are still mottled62,63.
Therefore, R is silenced in microgametophytes and the
activity is restored stochastically during epidermal
development, which results in mottled pigmentation.
Ploidy constraints, the requirement for paternal
transmission of some loci and the paternal downregula-
tion of R expression — all of which are observed in the
maize endosperm — have not been observed in the
maize embryo. For example, some R alleles that are sub-
ject to paternal imprinting are uniformly visibly
expressed in embryo structures62,63. These data have been
interpreted to mean that any paternal mark is readily
reversed after fertilization in the embryo but not in the
endosperm. Alternatively, there could be differential
imprinting of the two sperm in a pollen grain followed
by preferential fertilization; in a few species, sperm
dimorphism and preferential fertilization have been doc-
umented64. Arguing against this, using maize sperm that
were rendered dimorphic after non-disjunction of B
chromosomes (FIG. 4), Faure et al.65 showed that both
sperm (one without B chromosomes and one with two
B chromosomes) can fertilize the egg in vitro. However,
in vivo there is preferential fertilization of the egg by the
B-containing sperm, although this does not result from
an inability of one sperm to fuse with the egg.
Investigation of paternal gene expression in the maize
embryo indicates that paternally transmitted green fluo-
rescent protein (GFP) transgenes are expressed shortly
after in vitro fertilization66. The highly condensed chro-
matin of maize sperm begins to decondense immedi-
ately after fertilization, GFP RNA is detectable at 4 hours
and fluorescence is seen 6 hours after fertilization —
many hours before the first zygotic cell division.
Therefore, paternal alleles can be readily expressed in
the maize zygote, at least in vitro. Future experiments
in which a sperm is fused with a central cell might show
that imprinting has occurred.
The question of whether the paternal genome
undergoes similar early activation in semine as well as
in vitro has been difficult to answer. Studies of the
expression of many different genes in developing endo-
sperms and embryos of Arabidopsis have addressed this
question with conflicting results. Using allele-specific
reverse-transcription polymerase chain reaction (RT-
PCR) to differentiate maternally and paternally trans-
mitted alleles from different strains, Vielle-Cazada et al.
found that the zygotes and early embryos of Arabidopsis
contained transcripts for the maternal allele of two
genes, but that the paternal allele transcripts were not
detectable for several days53. Also, using β-glucuronidase
expression under the control of 18 different promoters
that are active in zygotes and early embryos, they con-
firmed that maternal expression was readily scored but
paternal expression was absent. These results were sur-
prising, because most loci that are required for embryo
development show classic Mendelian transmission.
Self-pollination of a strain that is heterozygous at an
embryo-lethal locus produces 25% dead embryos, not
the 50% lethality that is expected from a maternal
© 2003 Nature Publishing Group
REVIEWS
effect situation. In a few cases, such as emb30 (GNOM),
mutants have a weak, early maternal defect that embryos
recover from later in development53.
Weijers et al. were quick to provide evidence that
some paternally transmitted transgene markers could be
expressed in a two-cell embryo (although most of their
examples were from later stages) and argued that the
genetic data show that paternal alleles must be expressed
early in development67. These studies, and others that are
summarized in TABLE 1, show non-equivalence of alleles:
both are expressed, but with quantitative differences.
Differing results probably reflect different experimental
methods (assaying transcription only versus transcrip-
tion and translation of the test gene) and the use of vari-
ous developmental stages. Furthermore, transcript
detection after fertilization could reflect either de novo
transcription or transmission of stored mRNA. Whether
or not the entire paternal genome is silent immediately
after fertilization, it seems that the expression of mater-
nal and paternal alleles is not equivalent for many loci
during early development. The delay in full activation of
the paternal genome might parallel the delay in zygotic
gene activity in many animals — the plant version of
the mid-blastula transition25. The maternal genome
might escape this because the female gametes, unlike the
sperm cells, do not have condensed chromatin and are
more metabolically active.
Regulation of imprinting in Arabidopsis
The analysis of several maternal-effect lethal and loss-
of-DNA-methylation mutants in Arabidopsis has
greatly advanced understanding of the mechanisms
that underlie parental allele inequality and the complex-
ity of its regulation. FERTILIZATION INDEPENDENT
ENDOSPERM (FIE) mutants of Arabidopsis show a
striking phenotype: the diploid central cell can prolifer-
ate in the absence of fertilization to form an aberrant
endosperm68. So, in a mutant background, the usual
requirement for paternal and maternal contributions to
endosperm development are bypassed. FIE encodes a
WD group POLYCOMB protein that acts to repress endo-
sperm development68. If a FIE/fie heterozygous plant is
pollinated by wild-type pollen, half of the embryos
abort at the heart stage; in a reciprocal cross, all of the
embryos are viable. Therefore, the maternal fie allele
alone controls viability, and the paternal FIE allele is
either imprinted completely or its expression is too late to
restore normal endosperm development. A second locus,
MEDEA (MEA), which encodes a SET (Su(var)3–9;
enhancer of zeste; trithorax) domain polycomb protein,
is similarly required in the megagametophyte to sup-
press precocious endosperm development69,70. After
fertilization, only a maternally derived MEA allele suf-
fices to prevent embryo abortion71. Paternal MEA alleles
are not expressed in the endosperm, but are expressed in
embryo structures70,72, echoing the story of the R alleles
of maize. A third gene that might be subject to imprint-
ing is FERTILIZATION INDEPENDENT SEED2 (FIS2),
which encodes a zinc-finger protein and is also involved
in the suppression of central cell proliferation before
fertilization73.
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Table 1 | Parent-of-origin effects on gene expression in Arabidopsis

Gene Method Tissue Embryo stage* References
Whole Embryo Endosperm
MEA RT-PCR of whole seed M>P – – H, T, WS, M 70
RT-PCR of dissected seed – M=P M T 70
M=P M WS
– M=P M>P or M M
RT-PCR of whole siliques M – – G 72
In situ hybridization – – M – 72
GUS protein fusion M M PG 73
Rare P M,P ‡ in cyst EG
VPE RT-PCR of dissected seed – – M=P § WS 70
FIE RT-PCR of dissected seed – M=P M>P T 75
M=P None WS
M=P None M
GFP reporter – M M F–S 75
– None None G
– M,P‡ M,P‡ H
GUS protein fusion – M M EG 73
– M,P ‡ M,P ‡ H
FIS2 GUS protein fusion – M M PG, later § 73
SIN1 GUS reporter – M M EG 79
– None None LG–T
– M,P ‡ M,P ‡ WS
PRL RT-PCR of whole siliques M – – G 53
M,P ‡ – – T
GUS reporter – – M PG 53
– – M,P || MG
In situ hybridization to – M=P M>P O, H 89
GUS reporter
18 genes GUS reporter – M M PG–G § 53
EMB30/GN RT-PCR of whole siliques M – – PG 53
LTP1 2-component GUS reporter – M>P – H, T 67
– M – O 90
– M – G
– M,P ‡ – H
CYC 2-component GUS reporter – M – Z 90
– M# – O
– M,P ‡ – LG
RPS5A GUS reporter – M,P – PG, later 67
*Embryo stages (in order of development): Z, zygote; F, four-cell; O, octant; S, sixteen-cell; EG, early globular; MG, mid globular; LG, late
globular; H, heart; T, torpedo; WS, walking stick; M, mature. The stages before globular (G) are collectively referred to as pre-globular (PG).
‡
Maternal (M) and paternal (P) alleles were both expressed but no quantitative comparison was made. §Data not shown in the original
reference. || M everywhere, P in cyst only. #M expressed, P variable. CYC, CYCLOIDEA; EMB30/GN, EMBRYO DEFECTIVE 30/GNOM;
FIE, FERTILIZATION INDEPENDENT ENDOSPERM; FIS2, FERTILIZATION INDEPENDENT SEED 2; GFP, green flourescent protein; GUS,
β-glucuronidase; LTP1, LIPID TRANSFER PROTEIN 1; MEA, MEDEA; PRL, PROLIFERA; RPS5A, RIBOSOMAL PROTEIN S5A; RT-PCR,
reverse-transcription polymerase chain reaction; SIN1, SHORT INTEGUMENT 1; VPE, VACUOLAR PROCESSING ENZYME.

An important step in understanding MEA was the
recent discovery that a DNA glycosylase that is encoded
by DEMETER (DME) is expressed in the central cell
preceding fertilization. DME expression is required for
the activation of the megagametophyte copy of MEA,
which is consequently most strongly expressed in the
central cell74. After fertilization, DME transcripts are no
longer detected, although maternal MEA continues to
be expressed. No DME transcripts are detected in micro-
gametophytes, which indicates that one component of
the maternal requirement for MEA is the selective acti-
vation of an essential gene in the megagametophyte.
There are no detectable paternal MEA transcripts in the
endosperm, except in transgenic plants that ectopically
express DME after fertilization, in which the paternal
allele is presumably activated by the DME protein. At
present, MEA is the only known target of DME action.
In contrast to MEA, the expression of paternally
transmitted FIE alleles is detectable not only in the
embryo but also in the endosperm after an initial mater-
nal expression period75. So, although the FIE and MEA
proteins interact75,76, the duration (or severity) of pater-
nal lack of activity is distinctive. Furthermore, the
imprinting of these loci is differentially sensitive to loss
of function of the DECREASE IN DNA METHYLA-
TION (DDM1) gene. Maternal defects of mea mutants
can be rescued by ddm1 in two distinct ways. Seeds
inheriting mea can complete development if they are
also homozygous for ddm1 (REF. 72). Also, pollen from a
ddm1/ddm1 MEA/MEA plant suppresses maternal mea

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PHENOCOPY
A mimic of a phenotype that is
caused by a known mutation.
NATURE REVIEWS | GENETICS
defects75. One hypothesis is that lowered methylation
levels allow the expression of the paternal MEA allele
and that this is sufficient to result in normal develop-
ment; if so, paternal MEA should be independent of
activation by DME. Alternatively, reducing methylation
levels in sperm could allow the precocious expression of
paternal genes that are important in development, cir-
cumventing some MEA-mediated process. Interestingly,
a ddm1/ddm1 FIE pollen donor cannot relieve the defect
in fie maternal mutants75. A second experiment exploited
plants with low levels of DNA methyl transferase 1
activity, achieved using an antisense strategy. Paternal
FIE delivery from the antisense METHYL TRANS-
FERASE 1 (MET1) plants, which lack almost 90% of
normal DNA methylation levels, can rescue a fie1
maternal defect77. Also, pollination by MET1 antisense
plants rescues both maternal fis2 and mea defects, but,
unlike FIE, there is no requirement for a wild-type allele
from the pollen parent73.
Some caution is required in interpreting these results,
as different ecotypes and allele combinations have been
studied. Nonetheless, using genetic backgrounds with
altered levels of DNA methylation shows possible mech-
anistic differences between FIE and MEA or FIS2. The
available results indicate that the paternal hypomethyla-
tion of downstream targets can circumvent the require-
ment for MEA and FIS2, but not FIE. Unravelling the
suite of target genes and learning how their expression is
affected by maternal and paternal activity — or the lack
of it — remains a substantial challenge.
Significantly, the antisense MET1 plants show devel-
opmental defects similar to those observed in crosses
between diploid and hexaploid Arabidopsis 78. The polli-
nation of diploid females by MET1 antisense males
PHENOCOPIES crosses with excess maternal dosage. Con-
versely, the pollination of MET1 antisense females by
diploid males phenocopies crosses with excess paternal
dosage. Presumably, the demethylation of alleles in the
endosperm de-represses genes that are normally only
expressed from the opposite parent. A final known
player is SHORT INTEGUMENT 1 (SIN1), which
encodes a Dicer homologue79,80 and is therefore pre-
dicted to process RNA into short molecules that can
modulate gene expression post-transcriptionally by
RNA destruction, or transcriptionally by directing
chromatin remodelling. This gene acts in the sporo-
phyte before meiosis to modulate megagametophyte
development.
We can now return to the initial quandary of a model
in which paternal gene expression is generally repressed
but there is Mendelian transmission of most develop-
mental genes. The genetic results indicate that a paternal
allele suffices for a normal embryo, but does not prove,
or even require, that paternal alleles are routinely
expressed immediately after fertilization. Recall that
plant embryos can form from somatic cells and from
haploid microgametophytic cells in culture — cells that
must entirely reprogramme their gene expression.
Clearly, forming an embryo in these situations does not
require precisely imprinted maternal and paternal alleles,
neither does it require either fertilization event.
© 2003 Nature Publishing Group
REVIEWS
It seems possible that paternal alleles could be less
readily expressed initially and still suffice to organize an
embryo when they are eventually expressed hours or
days after fertilization. It is probable that not all of these
genes have parent-of-origin effects. Possible mechanisms
for parent-of-origin effects are: gametophytic expres-
sion, such as DME; prolonged silencing of the paternal
allele, such as R, MEA and FIS2; an early requirement
for the gene before general paternal genome expression;
or a combination of these mechanisms. Perhaps the
maternal effects of the absence of MEA, FIE and FIS2
proteins are readily recognized because they encode
components of the system that is responsible for dif-
ferential maternal and paternal allele activity. Also,
silencing of the paternal alleles of MEA and FIS2 per-
sists much longer than general paternal silencing in
Arabidopsis. Most loci that are expressed in the embryo
might, in fact, be imprinted in the sperm for reduced
initial activity, but also programmed to escape from this
downregulation early in embryo development.
The parental conflict theory and an extension of the
effective haploid phase are two distinct phenomena that
might have some overlapping elements. During the
growth phase of the maize endosperm, it is clear that
paternal alleles at many loci are required to attain a nor-
mal size, as reflected in the stringent requirement for the
ploidy ratio of maternal:paternal chromosomes. It
seems that maternal alleles have a dominant role imme-
diately after fertilization, whereas paternal alleles are
required later, with the consequence that both parents
contribute uniquely to foster normal endosperm and
embryo development.
Conclusions
The complexities of the flowering-plant life cycle have
developmental, genetic and evolutionary consequences
that generate fundamental differences between plants
and animals. The existence of a genetically active hap-
loid phase shows that plants accumulate fewer deleteri-
ous recessive alleles and might be more tolerant of DNA
transposons than are animals. Conversely, the absence
of a germline and the late switch of somatic lineages in
the plant shoot to floral development allow genetic
mutations to accumulate and be represented in the
gametes. Therefore, plants generate greater genetic
diversity, but, through haploid selection, impose more
stringent selection than animals. Despite this function
of the haploid phase, the diversity of structures and
pathways that are expressed in gametophytes has steadily
declined over evolutionary time.
It is plausible that, with the advent of double fertil-
ization in flowering plants, a supplementary mechanism
has evolved whereby parent-of-origin effects, including
imprinting and ploidy, can act as an extra quality check.
The delay in the full activation of the paternal genome
extends the effective haploid phase to early embryo
development. Also, flowering plants, like the mammals,
conduct a rigorous genome quality check after fertiliza-
tion, but do so primarily by assessing the ploidy level in
the endosperm — an accessory seed-restricted organ
that is produced by a second fertilization event. In many
VOLUME 4 | MAY 2003 | 3 7 7
REVIEWS
plant species, a normal endosperm is a prerequisite for
robust embryo development and seed success; therefore,
ploidy requirements and the imprinting of endosperm-
expressed alleles can act to ensure that plant sexual
reproduction only occurs if two sets of appropriately
programmed gametes are united. Given the present
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Online links
DATABASES
The following terms in this article are linked online to:
MaizeGDB: http://www.maizegdb.org
ig | R
TAIR: http://www.arabidopsis.org
DDM1 | DME | emb30 | FIE | FIS2 | MEA | SIN1
FURTHER INFORMATION
Virginia Walbot’s laboratory:
http://www.stanford.edu/%7Ewalbot
Zea mays Database (ZmDB): http://zmdb.iastate.edu
Access to this interactive links box is free online.
VOLUME 4 | MAY 2003 | 3 7 9