IN THIS ISSUE
Genetic and Epigenetic Regulation of Embryogenesis
Embryogenesis is a crucial period in the
development of eukaryotes. In most plants,
embryogenesis begins with an asymmetric
cell division that gives rise to a polar embryo
having a larger basal cell and a smaller
apical cell. The embryo proper develops from
the apical cell, and the basal cell develops
into the suspensor, which is attached to the
ovule and serves as a conduit for nutrient
transfer to the developing embryo. The early
stages of Arabidopsis embryo development
have stereotyped cell divisions. Cotyledons
then develop and serve to store nutrients for
the seedling after germination. During late
seed development, the seed desiccates
and remains dormant until conditions are
right for germination.
Cell division and differentiation during
these events follow highly regulated patterns
that are influenced by both genetic
and epigenetic mechanisms. Mammalian
genomes undergo genomic reprogramming
during embryogenesis involving global
changes in DNA methylation believed to
play an important role in developmental
regulation of gene expression. DNA methylation
is often associated with transcriptionally
inactive portions of the genome
(heterochromatin), and during early embryogenesis,
large portions of the genome
undergo a demethylation and subsequent
remethylation process that is thought to
contribute to chromatin decondensation
and transcriptional activation of genes essential
for embryo development (Li, 2002).
This might have the effect of providing
coarse control over gene expression, allowing
hundreds of genes specifically required
for embryogenesis to be switched on during
this critical period and to remain off during
other stages of the life cycle. On perhaps
a more fundamental level, RNA polymerase
II is at the heart of transcriptional machinery
that transcribes protein-coding genes into
mRNA. The RNA polymerase II machinery
contains .85 polypeptides within 10 subcomplexes
(Holstege et al., 1998), and the
fine control of transcription involves the
activity of these and many other interacting
factors.
Two articles in this issue of The Plant Cell
report on genetic and epigenetic aspects of
the regulation of gene expression during
embryogenesis. Xiao et al. (pages 805–
814) show that DNA methylation performed
by METHYLTRANSFERASE1 (MET1) influences
gene expression during embryogenesis
in Arabidopsis and is critical for
normal development of the embryo and
seed viability. In another article, Ding et al.
(pages 815–830) show that the pentatricopeptide
repeat (PPR) protein GLUTAMINERICH
PROTEIN23 (GRP23) is a nuclear
protein that interacts with RNA polymerase
II and likely functions in regulating gene
expression during early embryogenesis.
DNA METHYLATION PLAYS A VITAL
ROLE IN PLANT EMBRYOGENESIS
In mammals, cytosine methylation occurs
mainly atCpG sites(acytosineresiduelinked
on its 3# side to the 5# side of a guanine
residue) and is maintained by DNA methyltransferase1
(Dnmt1), which acts on
hemimethylated DNA (double-stranded DNA
methylated on only one strand, which
occurs following DNA replication). During
gametogenesis and embryogenesis, DNA
methylation is lost (on both strands of
double-stranded DNA) over a large portion
of the genome and later in embryo development
is reestablished by de novo
methyltransferases Dnmt3a and Dnmt3b,
which act on fully unmethylated DNA. Both
DNA Methylation in Arabidopsis Embryogenesis.
Wild-type (A) and met1 mutant (B) embryos 3 d after pollination.
The Plant Cell, Vol. 18, 781–784, April 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
maintenance and de novo methylation are
essential in animals, as mutations in either
the Dnmt1 or Dnmt3 gene are embryo lethal
(Lietal.,1992;Okanoetal.,1999).Imprinting,
or the differential expression of gene alleles
dependent on the parent of origin, depends
on heritable DNA methylation patterns
established during gametogenesis in animals,
and imprinted genes are protected
from demethylation in the global reprogramming
during embryogenesis (Li, 2002).
In plants, imprinting of certain genes occurs
in the endosperm and affects endosperm
development (reviewed in Gehring et al.,
2004).
Arabidopsis contains at least three classesofDNA
methyltransferase genes:MET1,
which is a homolog of Dnmt1 and encodes
the major CpG maintenance methyltransferase;
CHROMOMETHYLASE3 (CMT3),
which encodes a plant-specific DNA methyltransferase
that acts mainly at CpNpG and
CpNpN residues; and DOMAINS REARRANGED1
(DRM1) and DRM2, which share
homology with Dnmt3 and are thought to
function as the major de novo methyltransferases
in plants (Cao and Jacobsen, 2002;
Cao et al., 2003). Xiao et al. analyzed met1-6
mutants of Arabidopsis, which carry a putative
nullallele ofMET1. Homozygousmet1-6
mutant embryos were found to contain
a number of defects in cell division of both
suspensor and embryo cells beginning at
the earliest stages of embryogenesis (see
figure). Although the mutant plants produced
some nonviable seed in the first
generation, most of the seed was viable,
suggesting that DNA methylation patterns
play a limited role during embryogenesis
or that other DNA methyltransferases may
able to partially compensate for MET1 loss
of function. Analysis of met1 cmt3 double
mutant plants showed that these genes
have partially overlapping functions and
confirmed that DNA methylation is critical
for embryogenesis and seed viability, as the
double mutants showed synergistic effects
on seed viability and plant growth relative to
either of the single mutants.
Xiao et al. noticed that met1 mutant
embryos resembled mutants having defects
in establishing auxin gradients (described
in Friml et al., 2003) in that the
plane of cell divisions and apical-basal
polarity appeared to be disrupted. Friml
et al. (2003) used the auxin-responsive
promoter DR5rev linked to green fluorescent
protein (GFP) to show that establishing
and maintaining auxin gradients is
a critical process during early embryogenesis.
Xiao et al. found that DR5:GFP transgene
expression was evenly distributed in
met1 mutant embryos, in contrast with the
pattern indicative of an auxin gradient in the
wild type, suggesting that DNA methylation
may be important for setting up and/or
maintaining auxin gradients in the developing
embryo. They also examined the
expression of PIN1, which encodes an
auxin efflux carrier shown by Friml et al.
(2003) to be responsible for establishing
auxin gradients in early embryogenesis.
PIN1 expression was found to be relatively
evenly distributed throughout the developing
embryo in met1 mutants, in contrast
with the pattern observed in wild-type
embryos, suggesting that DNA methylation
may affect PIN1 expression. However,
DNA methylation (as measured using DNA
methylation–sensitive restriction endonucleases
and DNA gel blot analysis) could
not be detected in the coding, upstream, or
downstream regions of PIN1 in DNA isolated
from either met1 or wild-type seedlings,
leading the authors to conclude that
DNA methylation might affect PIN1 expression
indirectly.
The authors analyzed gene expression of
three other genes that play important roles
in specification of cell identity during
embryogenesis: YODA (YDA), which encodes
a mitogen-activated protein kinase
kinase kinase involved in specifying embryo
and suspensor cell identity (Lukowitz
et al., 2004), and WUSCHEL-RELATED
HOMEOBOX2 (WOX2) and WOX8, which
are expressed specifically in apical and
basal cell lineages, respectively, of developing
embryos and encode homeodomain
transcription factors that influence
cell division (Haecker et al., 2004). YDA
expression (as measured by steady state
mRNA levels) was enhanced, whereas the
expression of both WOX genes was reduced
in seed of met1-6 mutants relative to
the wild type, suggesting that DNA methylation
status affects the expressionofgenes
that influence cell identity during embryogenesis.
The authors examined methylation
status of the YDA gene and found that
it is methylated in wild-type plants, and
this is dependent on MET1 activity, as
mutations in MET1 resulted in loss of DNA
methylation at the locus. This might explain
why YDA expression was elevated in met1-6
mutant seeds compared with the wild type.
The reduction in WOX gene expression
was perhaps surprising, as DNA methylation
typically is associated with gene
silencing, and hypomethylation in the mutant
therefore would be expected to result
in enhanced gene expression. It has been
shown that DNA methylation enhances
transcription of mouse insulin growth factor2,
apparently by blocking the binding of
repressors to a specific intragenic region
(Murrell et al., 2001). This suggests that
DNA methylation exerts a degree of genespecific
control over gene expression during
embryogenesis. The methylation status of
the WOX genes was not examined, but it
will be of interest to determine how the
decrease in DNA methyltransferase activity
in the met1 mutant embryos leads to decreased
transcription of WOX genes.
The work of Xiao et al. shows that DNA
methylation is critical for embryogenesis in
Arabidopsis and is involved in regulating
gene expression affecting both auxin responsiveness
and embryo cell identity.
REGULATION OF RNA POLYMERASE II
IN EMBRYOGENESIS
Eukaryotic transcription of protein coding
sequences into mRNA is performed by
RNA polymerase II. The core of this enzyme
supercomplex is essentially the same for
the many thousands of genes that are transcribed,
and specificity for the regulation of
gene expression lies within the multitude of
interacting factors and subunits that influence
formation and binding of the complex
to gene promoter regions (Holstege et al.,
1998).Screening of embryo-defective (emb)
and seed coat mutants in Arabidopsis has
identified ;750 genes that are required for
normal seed development (McElver et al.,
2001; Tzafrir et al., 2003, 2004). Among an
initial set of 250 EMB genes, ;5% were
predicted to be transcription factors (Tzafrir
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782 The Plant Cell
et al., 2004). The function of most of these
genes remains unknown.
Yang et al. isolated an emb mutant from
a collection of enhancer trap mutants that
carry an Ac/Ds transposable element linked
to the b-glucuronidase reporter gene (see
Springer et al., 1995; Sundaresan et al., 1995).
The defect was traced to a single recessive
embryo-lethal mutation that caused an
arrest of embryo development at the early
globular stage (before the 16-cell dermatogen
stage). Many of the mutant embryos
exhibited aberrant divisions of the embryo
proper and suspensor cells, whereas early
endosperm development was unaffected.
This is one of only a few emb mutants characterized
that shows defects at this very
early stage of embryo development. The
mutation was found to be a loss-of-function
rather than enhancer insertion in a gene
designated GRP23, which encodes a polypeptide
with a leucine zipper domain, nine
PPRs at the N terminus, and a Gln-rich Cterminal
domain with an unusual Trp-GlnGln
(WQQ) repeat.
PPR proteins constitute one the largest
protein families in plants, with .400 members
in Arabidopsis. Evidence is emerging
that a number of plant PPR proteins are
RNA binding proteins involved in posttranscriptional
processes, such as mRNA
processing, often in mitochondria and
chloroplasts (Lurin et al., 2004). For example,
PPR proteins have been identified as
fertility restorers of cytoplasmic male sterility
in petunia (Bentolila et al., 2002) and
rice (Wang et al., 2006). The March issue of
The Plant Cell highlighted the work of Wang
et al. (2006), who showed that cytoplasmic
male sterility in Boro II rice is caused by
a cytotoxic peptide encoded by an aberrant
mitochondrial open reading frame, and
fertility restoration in this system depends
on PPR proteins that block production of
the peptide by cleavage or degradation of
its mRNA. Lurin et al. (2004) hypothesized
that PPR proteins function as sequencespecific
adaptors, a role that is consistent
with the large number of proteins in this
family.
Ding et al. show that GRP23 is a nuclear
PPR protein that interacts physically with
subunit III of RNA polymerase II via its
C-terminal WQQ domain in experiments
with both yeast and plant cells. The gene
is expressed at a relatively high level in
gametophytes (particularly pollen grains)
and young embryos and at low levels in
endosperm tissue and in actively dividing
cells in meristems of vegetative tissues.
GRP23 therefore exhibits clear differences
relative to other known PPR proteins,
which mostly have been characterized as
putative RNA binding proteins expressed
at low levels in all tissues and predicted to
be targeted to organelles (e.g., Small and
Peeters, 2000; Lurin et al., 2004).
Structural sequence features of the gene
suggest that GRP23 is a novel type of basic
domain leucine zipper (bZIP) protein, as it
has a bZIP domain unrelated to known bZIP
proteins in Arabidopsis. bZIP domains are
known to be involved in protein–protein
interactions, forming homodimers or heterodimers
via the zipper portion of the
domain, and to bind DNA in a sequencespecific
manner via the basic part of the
domain. PPR motifs are also predicted to be
sequence-specific RNA or DNA binding
domains, and Ding et al. hypothesize that
GRP23 may bind directly to DNA cisregulatory
elements through the basic region
of the bZIP domain or through the PPR
motifs. The interaction of the WQQ domain
with RNA polymerase III, together with gene
expression patterns and mutant phenotypes,
suggest that GRP23 may recruit
RNA polymerase II to control the expression
of genes essential during early embryogenesis.
It will be important to determine the
downstream targets of transcriptional control
by GRP23, the function of the bZIP
domain and PPR motif, and the possibility of
direct sequence-specific binding to DNA.
Nancy A. Eckardt
News and Reviews Editor
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