RESEARCH ARTICLE
113
Development 137, 113-122 (2010) doi:10.1242/dev.043174
The MED12-MED13 module of Mediator regulates the timing
of embryo patterning in Arabidopsis
C. Stewart Gillmor1, Mee Yeon Park1, Michael R. Smith1, Robert Pepitone2, Randall A. Kerstetter2
and R. Scott Poethig1,*
SUMMARY
The Arabidopsis embryo becomes patterned into central and peripheral domains during the first few days after fertilization. A
screen for mutants that affect this process identified two genes, GRAND CENTRAL (GCT) and CENTER CITY (CCT). Mutations in GCT
and CCT delay the specification of central and peripheral identity and the globular-to-heart transition, but have little or no effect
on the initial growth rate of the embryo. Mutant embryos eventually recover and undergo relatively normal patterning, albeit at
an inappropriate size. GCT and CCT were identified as the Arabidopsis orthologs of MED13 and MED12 – evolutionarily conserved
proteins that act in association with the Mediator complex to negatively regulate transcription. The predicted function of these
proteins combined with the effect of gct and cct on embryo development suggests that MED13 and MED12 regulate pattern
formation during Arabidopsis embryogenesis by transiently repressing a transcriptional program that interferes with this process.
Their mutant phenotype reveals the existence of a previously unknown temporal regulatory mechanism in plant embryogenesis.
INTRODUCTION
Plant embryos exhibit both apicobasal and central-peripheral (radial)
polarity. The shoot and root apical meristems (SAM and RAM,
respectively) form at opposite ends of the apicobasal axis and
contain stem cell populations that give rise to all postembryonic
tissue. Lateral organs such as cotyledons arise at the boundary of the
central and peripheral domains, and adopt the pattern present in the
radial axis of the embryo. Thus, genes that specify central identity
are also expressed on the adaxial (upper) side of developing
cotyledon primordia, whereas genes that specify peripheral identity
are expressed on the abaxial (lower) side. This continuity supports
the conclusion that the adaxial surface of a lateral organ is a
component of the central domain, whereas the abaxial side is a
component of the peripheral domain (Kerstetter et al., 2001;
McConnell et al., 2001).
Genes involved in the specification of radial polarity were initially
identified in screens for mutations that affect the adaxial-abaxial
polarity of leaves and floral organs. In Arabidopsis, these screens
have produced mutations in three families of transcription factors.
Members of the class III HD-ZIP family of transcription factors
promote adaxial identity (McConnell et al., 2001; Emery et al.,
2003), whereas KANADI (KAN) and YABBY (YAB) transcription
factor families promote peripheral and abaxial identity (Kerstetter
et al., 2001; Eshed et al., 2001; Siegfried et al., 1999). Evidence that
these genes are also important for embryonic development is
provided by the phenotype of gain-of-function alleles, and the
phenotype of embryos lacking more than one member of these gene
families. For example, mutations that cause the HD-ZIP III gene
PHB to be ectopically expressed in the peripheral-abaxial domain
produce SAMs (central structures) on the abaxial side of leaves
1
Department of Biological Sciences, University of Pennsylvania, Philadelphia, PA
19104, USA. 2Department of Plant Biology and Pathology, The Waksman Institute,
Rutgers University, Piscataway, NJ 08854, USA.
*Author for correspondence ([email protected])
Accepted 9 October 2009
(McConnell and Barton, 1998). Conversely, phb phv rev triple
mutants lack a SAM (Emery et al., 2003). Ectopic expression of
KAN1 in the central domain of the embryo blocks the development
of the SAM, whereas kan1 kan2 kan4 triple mutant embryos have
outgrowths on the abaxial surface of cotyledons and the periphery
of the hypocotyl that have characteristics of leaf primordia
(Kerstetter et al., 2001; Izhaki and Bowman, 2007).
The studies described above have begun to address the
developmental role of KAN genes in embryonic and postembryonic
development. However, the mechanism responsible for the initial
establishment of radial polarity in embryogenesis remains a mystery.
To identify factors in the polarity pathway that act upstream of KAN
genes, we took advantage of an enhancer trap insertion in KAN2 that
expresses green fluorescent protein (GFP) in the peripheral-abaxial
domain of both embryonic and postembryonic organs. A screen for
mutations that affect the expression of this reporter produced alleles
of two genes, GRAND CENTRAL (GCT) and CENTER CITY (CCT),
which we found encode the Arabidopsis homologs of MED13 and
MED12.
In yeasts and animals, Med13 and Med12 act as transcriptional
repressors by inhibiting core Mediator, a multisubunit complex that
allows transcription factors bound at upstream enhancer elements to
activate RNA polymerase II. Med13 and Med12 have identical
mutant phenotypes in each organism (Samuelsen et al., 2003; Yoda
et al., 2005; Janody et al., 2003), indicating that they have similar
biological functions. In humans, MED13 negatively regulates
transcription by causing an allosteric change in core Mediator that
prevents its association with RNA polymerase II (Knuesel et al.,
2009), whereas MED12 recruits the histone methyltransferase G9a
to core Mediator, promoting epigenetic silencing of target genes via
methylation of chromatin H3K9 (Ding et al., 2008). In yeasts and
Drosophila, Med13 and Med12 physically interact with Cyclindependent kinase 8 (Cdk8) and Cyclin C (CycC) to form a complex
known as the Cdk8 module of Mediator (Samuelsen et al., 2003;
Loncle et al., 2007). However, the observation that in Drosophila
Cdk8 and CycC do not have the same mutant phenotype as Med13
(skd – FlyBase) and Med12 (kto – FlyBase), implies that these
DEVELOPMENT
KEY WORDS: Arabidopsis, Embryo, Heterochrony, MEDIATOR, MED12, MED13, Polarity, KANADI
RESEARCH ARTICLE
components are functionally distinct (Loncle et al., 2007). In both
Drosophila and Caenorhabditis elegans, Med13 and Med12 specify
cell identity by regulating downstream targets of the Wnt signaling
pathway (Janody et al., 2003; Yoda et al., 2005; Carrera et al., 2008).
In particular, the Drosophila Med13 and Med12 genes skuld and
kohtalo control the cell affinities that maintain boundaries between
anteroposterior and dorsoventral compartment boundaries of
the wing disc (Janody et al., 2003; Loncle et al., 2007). The
developmentally specific phenotypes of mutations in Med13 and
Med12 in Drosophila and C. elegans are consistent with a
microarray analysis of these genes in yeast, which indicates that they
regulate a relatively small number of genes (Samuelsen et al., 2003).
This is in contrast to core Mediator, which is required for nearly all
transcription (Kornberg, 2005).
The core Mediator complex was recently purified from
Arabidopsis suspension culture cells (Bäckström et al., 2007). The
purified complex contained most of the components present in the
core complex in other organisms, but was missing proteins in the
Cdk8 module, consistent with the limited and transient interactions
observed between these proteins and core Mediator in other systems
(Andrau et al., 2006). The phenotype of mutations in two
components of the core complex (PFT1/MED25 and SWP/MED14)
has been described in Arabidopsis. Mutations of PFT1/MED25
delay flowering under suboptimal light conditions (Cerdán and
Chory, 2003; Bäckström et al., 2007), whereas mutations of
SWP/MED14 cause a premature arrest in cell proliferation during
vegetative growth, resulting in extreme dwarfing (Autran et al.,
2002). By contrast, mutations in the Arabidopsis Cdk8 homolog
HEN3 have a mild reduction in cell expansion in leaves, and only
show more severe phenotypes in combination with mutations that
affect pre-mRNA processing (Wang and Chen, 2004).
Here we show that MED13 and MED12 regulate developmental
patterning in Arabidopsis. We found that MED13, corresponding to
the GCT gene, and MED12, corresponding to the CCT gene, are
absolutely required for KAN expression during early embryogenesis,
and promote KAN expression during postembryonic development
as well. Analysis of markers for the SAM and vascular tissue
showed that gct and cct mutations have a unique effect on embryo
patterning: the initiation of a number of patterning genes is delayed
for several days, after which they are expressed in an almost normal
manner. The expression pattern and proposed biochemical function
of GCT and CCT is consistent with a role for these genes in
transiently repressing transcription of genes that would otherwise
interfere with radial pattern formation. Our results reveal a role for
MED12 and MED13 in the regulation of developmental timing, and
uncover a novel temporal component of pattern formation in
Arabidopsis embryogenesis.
MATERIALS AND METHODS
Genetic stocks and growth conditions
All seed stocks are in the Columbia ecotype. Enhancer trap lines E2023 and
E2331 were generated using the pBIN 35S mGAL4-VP16+UAS mGFP5ER vector, containing a minimal promoter driving ER localized mGFP5
(Haseloff, 1999). E2023 contains at least two copies of the GAL4UAS::GFP enhancer trap in inverted orientation in the second intron of the
KAN2 gene (at bp 2889 of the genomic sequence relative to start codon), as
well as a second linked T-DNA insertion between genes At1g29880 and
At1g29890. The enhancer sequences driving E2331 expression have not
been defined. The kan1-12 allele (Kerstetter et al., 2001) and the kan2-4
allele (SALK_095643) (Wu et al., 2008) were used for double mutant
analysis. Plants were grown under 16 hours fluorescent illumination (100
E/m2; Sylvania VHO) at 22°C in Farfard #2 soil. SALK T-DNA insertion
lines (Alonso et al., 2003) and SAIL T-DNA insertion lines (Sessions et al.,
Development 137 (1)
2002) were obtained from the Arabidopsis Stock Center (ABRC) at
www.arabidopsis.org. Seed for gct-2, cct-1, E2023, E2331 and gPHB-GUS
have been deposited in the Arabidopsis Stock Center (ABRC).
E2023 seeds were mutagenized as follows: 100 mg of seed was
suspended in 25 ml of water with 3 l Tween-20 for 15 minutes, rinsed and
then soaked overnight in water at 4°C on a rotator. Seeds were then
suspended in 10 ml of 0.4% ethyl methane sulfonate in 100 mM sodium
phosphate buffer (pH 7) for 9 hours on a rotator at room temperature, and
subsequently rinsed 10 times with 25 ml of water. Embryos from three
consecutive siliques on the main inflorescence of 2500 M1 plants were
examined for changes in E2023 expression and embryo morphology. The
main inflorescence of M1 plants was consistently found to have two or three
sectors, and thus our screen comprised 5000-7500 individual mutagenized
sectors. Mutant lines were backcrossed three times before analysis.
Molecular biology
RNA in situ hybridization was performed using a protocol obtained from
Jeff Long (www.its.caltech.edu/~plantlab/protocols/insitu.pdf), with the
following minor modifications. The probe was hybridized to slides at a
temperature of 60-65°C overnight. The blocking and antibody dilution
solutions were produced using maleic acid instead of Tris-HCl. For the STM,
GCT and CCT probes, partial cDNA sequences were amplified and
transcribed using the following primers: STM-T7, 5⬘-CAAGCTTAATACGACTCACTATAGGGAAAGGATTGCCCAAGACAT-3⬘; STMSP6, 5⬘-CTCTAGATTTAGGTGACACTATAGGATCCATCATAACGAAATCGT-3⬘; GCT-SP6, 5⬘-ATTTAGGTGACACTATAGAAGCCCCAAATTAGTTCTACAAGTGG-3⬘;
GCT-T7,
5⬘-TAATACGACTCACTATAGGGAGACCAGGTCCAGAACCTCTGC-3⬘; CCT-SP6,
5⬘-ATTTAGGTGACACTATAGAAGAAGCTTGTATCTGTATTAGATTGC-3⬘; CCT-T7, 5⬘-TAATACGACTCACTATAGGGAGACCTTCCTCAATCACTTCCCC-3⬘.
The gPHB-GUS translational fusion construct was made by cloning
10443 bp upstream of the PHB stop codon in front of the GUS+ gene in the
pCAMBIA3300 vector, with an NAAIRS linker replacing the PHB stop
codon. This DNA fragment contains all sequences from the previous gene
on the 5⬘ end of PHB until the PHB stop codon, including all introns and the
miR165/166 target sequence. Plants homozygous for the gPHB-GUS
construct show mild adaxialized phenotypes, indicating that the PHB-GUS
fusion protein is biologically active.
Microscopy and histology
The E2023 genetic screen was conducted using a Leica MZFLIII
fluorescence microscope equipped with a long pass GFP filter. To detect
GUS activity, embryos were stained at 37°C for 1h (pKAN::GUS,
pSTM::GUS) or overnight (gPHB-GUS) according to Donnelly et al.
(Donnelly et al., 1999). Embryos were cleared in Hoyer’s solution (bent
cotyledon stage embryos) or mounted in 5% glycerol (globular to torpedo
stage) and examined with DIC optics. Confocal images were obtained using
a Leica TCS SL confocal microscope with a 20⫻ water immersion lens.
Embryos were removed from ovules using DuMont #5 forceps and mounted
in 1⫻MS salts plus 500 mM glucose. Globular and heart stage embryos
were counterstained by adding Nile Red to MS-glucose to a final
concentration of 3 g/ml (Jenik et al., 2005). For camera lucida drawings,
wild-type and mutant leaves were vacuum infiltrated with 1% Tween 20 in
water, and manually sketched using an Olympus BX40 microscope
equipped with a U-DA light tube and mirror.
RESULTS
A genetic screen to identify upstream regulators
of peripheral-abaxial polarity
The KAN family of transcription factors specify peripheral-abaxial
identity beginning in early embryogenesis (Kerstetter et al., 2001;
Eshed et al., 2004). To identify genes that act upstream of KAN in
the embryo polarity pathway, we took advantage of the enhancer
trap line E2023, which contains a GAL4-UAS::GFP insertion
(Haseloff, 1999) that disrupts the KAN2 gene (see Materials and
methods). This line expresses GFP strongly and constitutively
DEVELOPMENT
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Temporal control of embryo patterning
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115
Fig. 1. Mutations in GRAND CENTRAL (GCT) and CENTER CITY (CCT) cause decreased expression of the peripheral-abaxial enhancer
trap E2023. (A-F)E2023 is a KAN2 reporter expressed in the peripheral-abaxial domain of wild-type plants. Early heart (A), late heart (B), torpedo
(C) and bent cotyledon (D) embryos; inset in D shows embryo cross section. Abaxial view of a 10-day-old seedling (E) reveals that E2023 is
expressed in abaxial vascular tissue of expanded cotyledons and leaves, and throughout the abaxial side of leaf primorida (arrowhead). Carpels of
wild-type flower (F). gct (G-L) and cct (M-R) delay and reduce the expression of E2023. Early heart (G,M), late heart (H,N), torpedo (I,O) and bent
cotyledon (J,P) stage embryos. Ten-day-old gct (K) and cct (Q) seedlings (insets in K and Q: 2⫻ magnification of leaves). gct (L) and cct (R) carpels.
Scale bars: 20m in A-D,G-J,M-P; 1 mm in E-F,K-L,Q-R.
significant phenotypic differences were noted between the various
alleles of gct; gct-2 and cct-1 (hereafter referred to as gct and cct)
were used for all of the experiments presented here. Homozygous
plants produce very few seeds, and thus gct and cct embryos were
obtained from heterozygous plants, allowing us to stage the
development of mutant embryos by observing wild-type embryos
segregating in the same silique. Thus, for all analyses in this
manuscript, the developmental stage of mutant embryos was
inferred from wild-type siblings of the same chronological age.
GCT and CCT also regulate the expression of E2023 during
postembryonic development, and affect the timing of a variety of
developmental transitions including the embryo-to-seedling
transition, vegetative phase change and flowering time. This
paper focuses primarily on their role in embryogenesis.
GCT and CCT are absolutely required for E2023 expression at the
heart stage of embryogenesis, and promote the correct level of
expression later in embryo development (Fig. 1G-J,M-P). E2023 is
undetectable in gct embryos (Fig. 1G-I) until the bent cotyledon
Fig. 2. gct enhances the polarity phenotypes of kanadi mutants. (A-D)Wild-type (A), kan1 kan2 (B), gct (C) and gct kan1 kan2 (D) embryos
at the early bent cotyledon stage of development. (E-H)Four-week-old wild-type (E) and kan1 (F) plants with inflorescences removed, and 5-weekold gct (G) and gct kan1 (H) plants. (I-L)Camera lucida drawings of adaxial and abaxial mesophyll tissue of leaf 1 of wild-type (I), kan1 (J), gct (K)
and gct kan1 (L). (M-P)Wild-type (M), kan1 (N), gct (O) and gct kan1 (P) flowers. The arrowheads in D and P point to sites of ectopic outgrowths.
The dotted lines in M-P underline stigmatic tissue. The leaves in E, F and G are numbered according to the order of their initiation. Scale bars:
20m in A-D; 1 cm in E-H; 2 mm in M-P.
DEVELOPMENT
within the peripheral-abaxial domain of embryos, the abaxial
vasculature of leaves and the abaxial domain of carpels (Fig. 1A-F),
an expression pattern that is essentially identical to that of KAN2
(Emery et al., 2003; Eshed et al., 2004). It has no obvious
morphological defect beyond the production of a small longitudinal
ridge of tissue below the first bract, and an occasional flower with
five petals (data not shown).
Examination of approximately 6000 mutagenized M1 sectors
(see Materials and methods) yielded more than 50 mutant lines
with a heritable effect on E2023 expression. Five of these lines
segregated embryos with a nearly identical recessive mutant
phenotype, consisting of a decrease in the intensity of E2023
expression (Fig. 1G-J,M-P), and an increase in size of the SAM
(see below). Complementation analysis revealed that these
mutations represented two genes, which were named GRAND
CENTRAL (GCT) (four alleles) and CENTER CITY (CCT) (one
allele), to reflect the increased size of the SAM and the decrease
in expression of the peripheral-abaxial enhancer trap E2023. No
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Development 137 (1)
stage (Fig. 1J), and is weakly expressed beginning at the torpedo
stage in cct embryos (Fig. 1O). At the bent cotyledon stage, its
expression is confined to patches on the abaxial side of the
cotyledons in both mutants (Fig. 1J,P). After germination, gct and
cct express E2023 in leaves in the same spatial pattern as wild-type
plants, but at a reduced level (Fig. 1K,Q). E2023 expression was not
detectable in carpels of gct and cct plants (Fig. 1L,R). Plants
heterozygous for both gct and cct segregated the same range of
embryo phenotypes as plants heterozygous for gct or cct only,
implying that double mutant embryos have essentially the same
phenotype as single mutants. Although we did not attempt to
identify double mutants during embryogenesis, plants homozygous
for both gct and cct were identified after germination using
molecular markers, and supported this conclusion. This result
suggests that GCT and CCT have closely related functions and may
act in the same pathway.
Fig. 3. GCT and CCT promote KAN1 expression in early
embryogenesis. (A-D)gPHB-GUS is expressed in the SAM and vascular
primodia of wild-type transition stage embryos (A), on the adaxial side of
cotyledons and in vascular primordia of wild-type heart stage embryos
(B), in vascular primordia and the adaxial epidermis of cotyledons in wildtype torpedo stage embryos (C) and in the SAM of bent cotyledon
embryos (D). A similar pattern is observed in gct (E-H) and cct (I-L)
embryos, with the exception that gPHB-GUS is not expressed in the
adaxial epidermis of cotyledons. (M-O)A pFIL::dsRed-N7 transcriptional
fusion is expressed on the abaxial face of cotyledons in torpedo stage
wild-type (M) gct (N) and cct (O) embryos. (P-R)Expression of a
pKAN1::GUS transcriptional fusion in the peripheral domain of wild-type
embryos at the heart (P), torpedo (Q) and bent cotyledon stages (R). No
expression of pKAN1::GUS in heart stage gct (S) and cct (V) embryos,
initiation of expression in torpedo stage gct (T) and cct (W) embryos, and
reduced but spatially normal expression in wild-type bent cotyledon stage
gct (U) and cct (X) embryos. Scale bar: 20m.
GCT and CCT regulate KAN expression
independently of PHB
To further characterize the role of GCT and CCT in the specification
of adaxial-abaxial polarity, we examined the effect of gct and cct
mutations on expression of a representative member of the KAN, YAB
and class III HD-ZIP gene families. The pKAN1::GUS construct (Wu
et al., 2008) expresses GUS in the peripheral domain of the hypocotyl
throughout embryogenesis (Fig. 3P-R). gct and cct delayed the
expression of pKAN1::GUS until the torpedo stage of development
(Fig. 3S-T,V-W) and slightly reduced its level of expression, but by
the bent cotyledon stage had no obvious effect on its spatial expression
DEVELOPMENT
gct enhances polarity defects of kanadi mutants
KAN1, KAN2 and KAN4 have overlapping functions in the
specification of abaxial identity, and produce strong effects on
embryo polarity only when all three genes are absent (Izhaki and
Bowman, 2007). The observation that gct and cct reduce the
expression of E2023 (KAN2) but do not have a polarity phenotype
as severe as the kan1 kan2 kan4 triple mutant, suggests that GCT and
CCT may not completely abolish KAN gene expression during
embryogenesis, or may affect the expression of some KAN genes but
not others. Reasoning that this effect might be visible only in a
sensitized genetic background, we characterized the phenotype of
gct in combination with kan1 and kan1 kan2.
gct enhanced the phenotype of kan mutations at every stage of
plant development we examined. The embryonic phenotype of
kan1kan2 double mutants is similar to that of gct (Fig. 2A-C). Like
gct (Fig. 2C), kan1kan2 embryos (Fig. 2B) display a slight delay in
morphogenesis, and have cotyledons that splay outward, a
phenotype typical of a loss of abaxial identity. gct kan1kan2 triple
mutants resemble gct, but differ from gct in having ectopic growths
on both the cotyledons and hypocotyl (arrowheads, Fig. 2D). These
ectopic growths have previously been observed only in kan1 kan2
kan4 triple mutants (Izhaki and Bowman, 2007).
gct also enhanced kan1 postembryonic phenotypes. Whereas
the leaves of kan1 and gct are held horizontally (Fig. 2E-G), the
leaves of gct kan1 double mutants stand upright, curl upwards and
are irregularly expanded (Fig. 2H). This effect on leaf
morphology is associated with a change in polarity of mesophyll
cells. Adaxial (palisade) mesophyll cells are typically round and
densely packed, whereas abaxial (spongy) mesophyll cells are
irregular in shape and are separated by large air spaces (Fig. 2I).
kan1 (Fig. 2J), and to a lesser extent gct (Fig. 2K), cause spongy
mesophyll cells to be less convoluted and more highly packed
than normal. The spongy mesophyll of gct kan1 plants (Fig. 2L)
shows an enhanced loss of abaxial identity, consisting of more
densely packed, less branched cells that have a morphology
intermediate between wild-type adaxial and abaxial mesophyll
cells. Although kan1 and gct do not have a major effect on carpel
development (Fig. 2M-O), gct kan1 flowers (Fig. 2P) have an
enlarged septum with increased stigmatic tissue, less valve tissue
and abaxial projections of carpel and papillary tissue, and are
essentially identical to the flowers of kan1 kan2/+ plants (Pekker
et al., 2005). These results are consistent with the decreased
expression of E2023 in gct, and suggest that GCT contributes to
the specification of peripheral-abaxial identity by promoting the
expression of KAN2.
Temporal control of embryo patterning
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117
pattern (Fig. 3R,U,X). Thus, gct and cct have the same effect on the
expression of KAN1 as they have on the expression of KAN2 (Fig. 1GJ,M-P). By contrast, gct and cct did not affect the expression of
pFIL::dsRed-N7, a reporter for the abaxially expressed gene
YABBY1/FILAMENTOUS FLOWER (FIL) (Heisler et al., 2005) (Fig.
3M-O; see Fig. S1 in the supplementary material).
Class III HD-ZIP genes interact antagonistically with KAN genes
(Eshed et al., 2004). To determine if the decrease in KAN1 and KAN2
expression in gct and cct embryos is the result of an increase in HDZIPIII expression, we examined their effect on expression of the
HD-ZIPIII gene PHB. Because PHB is regulated posttranscriptionally by miR165/miR166, this experiment was
conducted using a reporter in which GUS was fused to the Cterminal end of a PHB genomic clone (gPHB-GUS). This construct
has an expression pattern identical to that reported for PHB mRNA
(McConnell et al., 2001) (Fig. 3A-D). Interestingly, although gct and
cct reduced gPHB-GUS expression on the adaxial side of
cotyledons, the timing and overall GUS pattern in these mutants was
essentially normal. Thus, the decrease in KAN expression in gct and
cct is not attributable to an increase in the size of the PHB expression
domain, or to an increase in the level of PHB expression.
phenotype of gct-2 (Fig. 4A). CCT is located at the top of
chromosome 4 and corresponds to At4g00450; cct-1 is a missense
mutation in At4g00450, and two T-DNA insertions in At4g00450
(SALK_108241 and SALK_124276) have a phenotype identical to
cct-1 and failed to complement this point mutation (Fig. 4B).
GCT and CCT are single copy genes in Arabidopsis. The predicted
GCT protein has 1921 amino acids (AA), and contains a serine-rich
region between AA 407 and 426. gct-3 changes the first serine of this
domain to an asparagine, suggesting that this domain is functionally
important. AA 1088-1179 of the predicted GCT protein are 42%
identical to the MED13 pfam06333 domain (Fig. 4A). The entire
predicted GCT protein is 16% identical and 19% similar to human
MED13, and regions of identity and similarity are distributed
throughout the protein (see Fig. S2 in the supplementary material).
The predicted CCT protein is 2144 AA long; AA 69-127 are 37%
identical to the MED12 pfam09497 domain (Fig. 4B). The entire
predicted CCT protein is 12% identical and 21% similar to human
MED12 (NP_056150), and regions of identity and similarity are
distributed throughout the protein (see Fig. S3 in the supplementary
material). These results suggest that the MED12/MED13 regulatory
module of Mediator is conserved in plants.
GCT and CCT encode MED13 and MED12, predicted
transcriptional repressors conserved in all
eukaryotes
The molecular identities of GCT and CCT were determined using a
map-based approach (Lukowitz et al., 2000). GCT is located on the
lower arm of chromosome 1 and corresponds to At1g55325; all four
EMS-induced gct alleles contained mutations in At1g55325, and a
T-DNA insertion in At1g55325 (SAIL_1169_H11) has a phenotype
identical to these EMS alleles and failed to complement the mutant
GCT and CCT are dynamically expressed during
embryo development
The expression of GCT and CCT during embryogenesis was
characterized by RNA in situ hybridization. GCT mRNA was
detected in both the apical and basal cell at the one-cell embryo stage
(Fig. 4C), whereas CCT was first detected at the dermatogen stage
of embryogenesis (Fig. 4L). Both genes are expressed in all cells of
the embryo proper and suspensor through the globular stage of
embryogenesis (Fig. 4C-F,L-M), but their expression begins to
DEVELOPMENT
Fig. 4. GCT and CCT encode the Arabidopsis
homologs of the transcriptional regulators MED13
and MED12, and are dynamically expressed during
embryogenesis. (A)GCT corresponds to At1g55325. The
location and the nature of mutant alleles are shown.
Serine-rich region (AA 407-426) shown in dark gray.
Region of 42% identity (P-value 9e–11) to pfam06333
MED13 domain shown in light gray (AA 1088-1179).
(B)CCT corresponds to At4g00450. Region of 37%
identity (P-value 5e–13) to pfam09497 MED12 domain
(AA 69-127) is shown in light gray. (C-K)mRNA in situ
hybridization analysis of GCT expression in (C) one-cell, (D)
octant, (E) dermatogen, (F) globular, (G) heart, (H) torpedo,
(I) bent cotyledon, (J) SAM of bent cotyledon, and (K) RAM
of bent cotyledon stage embryos. (L-R)RNA in situ
hybridization analysis of CCT expression in (L) dermatogen,
(M) globular, (N) heart, (O) torpedo, (P) bent cotyledon,
(Q) SAM of bent cotyledon and (R) RAM of bent cotyledon
stage embryos. (S)GCT sense probe; (T) CCT sense probe.
Scale bar: 10m.
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Development 137 (1)
decrease in the periphery of the hypocotyl and on the abaxial side of
cotyledons starting at the heart stage (Fig. 4G,N). This trend
continues through the torpedo stage (Fig. 4H,O), and by the bent
cotyledon stage GCT and CCT mRNAs are restricted to the vascular
tissue and SAM and RAM (Fig. 4I-K,P-R). This pattern is consistent
with the temporal effect of gct and cct mutations on the expression
of E2023 and pKAN1::GUS (Figs 1 and 3), and suggests that these
genes are important in the peripheral domain of the embryo only
early in embryogenesis. Postembryonically, public microarray
expression data indicate that GCT and CCT are expressed in most
tissue types, with highest expression in the shoot apex (Winter et al.,
2007).
gct and cct uncouple cell division, pattern
formation, and morphogenesis
Our analysis of GCT and CCT expression demonstrated that their
mRNAs are present in wild-type embryos before the initiation of
KAN expression. To explore additional roles for GCT and CCT in
early embryogenesis, the histological phenotype of wild-type, gct
and cct embryos was characterized from the earliest stages of
development. We found that pattern formation and morphogenesis
are delayed in gct and cct embryos compared with wild type (Fig.
5A-K).
In both gct and cct the first division of the apical cell is transverse
to the long axis of the embryo, instead of parallel as in wild type
(arrows, Fig. 5A,B); furthermore, cells of the embryo proper
(brackets) are abnormally elongated, and in this respect resemble
suspensor cells (marked by the dashed line). gct (Fig. 5D) and cct
(Fig. 5E) globular embryos are more elongated than wild type (Fig.
5C), and have more suspensor cells. The more severely affected
embryos also lack the lens-shaped cell produced by asymmetric
division of the uppermost cell of the suspensor (asterisks in Fig.
5D,E), and have one or more extra tiers of cells between the
suspensor and the lower tier of the embryo (‘x’ in Fig. 5D,E). These
extra tiers probably result from the abnormal first division plane of
the apical cell shown in Fig. 5A,B. Although the percentage of
phenotypically mutant gct and cct embryos segregating in siliques
of heterozygous plants is quite low at the octant, dermatogen and
globular stages (approximately 5%), these phenotypes were never
observed in octant (n227), dermatogen (n215) or globular stage
(n344) embryos from wild-type plants.
In wild-type embryogenesis, the onset of the heart stage of
development is marked by the initiation of cotyledon primordia
on the upper flanks of the embryo, and the appearance of
elongated vascular cells (Fig. 5F). gct and cct delay both of these
patterning events (Fig. 5G,H). The most strongly affected mutants
DEVELOPMENT
Fig. 5. gct and cct mutants uncouple pattern
formation, growth and morphogenesis in early
embryos. (A,B)Wild-type and gct embryos at the octant
stage (A), and wild-type and cct embryos at the
dermatogen stage (B). The plane of the first division is
marked with an arrow, the embryo is marked with a
bracket and the suspensor is marked with a dashed line.
(C)Wild-type globular stage embryo. (D)Weak (left) and
strong (right) globular stage gct embryos. (E)Globular
stage cct embryos show similar defects. In B-E, the cells of
the upper tier and their derivatives (u), the lower tier and
their derivatives (t), extra tiers in gct and cct (x) and the
uppermost cell of the suspensor and its derivatives
(asterisks) are labeled. (F)Wild-type early heart stage
embryo. (G)Early heart stage gct embryos with two (left)
or one (middle) small cotyledon primordia, or with
random divisions throughout the embryo (right).
(H)Similar phenotypes are seen in early heart stage cct
embryos. (I-K)At the wild-type late heart stage (I), gct (J,
left) and cct (K, left) embryos with two cotyledon
primordia, and gct (J, right) and cct (K, right) embryos
with random divisions throughout the embryo. In F-K, the
shoot apical meristem, root apical meristem, and vascular
primordia (vp) are indicated with lines, and the cotyledon
primordia with arrowheads. (L)Cell numbers in medial
sections of wild-type, gct and cct embryos at the early
globular, late globular, early heart and late heart stages.
Standard deviation is shown at top of bars in L (n≥8). All
images in A-K are tracings of cleared embryos. At each
developmental stage, the percentage of each phenotypic
class of mutant embryos in siliques of heterozygous plants
is shown, along with the total number of embryos scored.
All images shown at equal magnification. Scale bar:
10m.
Temporal control of embryo patterning
RESEARCH ARTICLE
119
Fig. 6. SAM and RAM defects in late stage gct and cct embryos. (A-E)Bent cotyledon stage wild-type, gct and cct embryos: 86% (n151) of
late stage gct embryos have an enlarged SAM (B); 14% (n151) of late stage gct embryos lack the small vacuolated cells and layered tissue
organization characteristic of the SAM (C); 94% (n171) of late stage cct embryos have an enlarged SAM (D); 6% (n171) of late stage cct
embryos lack a SAM (E). (F)Medial section of wild-type SAM contains an average of 25±1 cells, (n15). (G)Medial section of enlarged gct SAM
contains an average of 68±5 cells (n15). (H)gct embryo with large vacuolated cells in place of the SAM. (I)Medial section of enlarged cct SAM
contains an average of 66±8 cells (n15). (J)cct embryo with large vacuolated cells in place of the SAM. (K)Medial optical section of a wild-type
RAM. (L)Of gct embryos, 86% had a slightly disorganized root cap and extra cells in RAM (n84). (M)In addition, 14% of gct embryos lacked most
cells of the root cap (n84). (N)Of cct embryos, 100% (n81) had a slightly disorganized root cap and extra cells in RAM. (O-Q)Hypocotyl of wildtype (O), gct (P) and cct (Q) embryos. Percentages in B-E refer to mutant embryos only, which are easily distinguished at late stages by their
meristem phenotype. In A-E, the location of the SAM and RAM are shown in boxes. Original cleared images and tracings of the SAM, RAM or
hypocotyl are shown in F-Q. The SAM was defined as the region bounded by vascular tissue. A-E and F-Q shown at equal magnification. Scale bars:
40m in A; 10m in F. Asterisks indicate quiescent center cells of root meristem. co, cortex; en, endodermis; ep, epidermis; lrc, lateral root cap; p,
pericycle; v, vascular tissue; x, extra RAM cells.
of the RAM and root cap, but to a lesser extent than the SAM (Fig.
6K-N). gct and cct increase the size of hypocotyl cells, but do not
change the number of tissue layers in the hypocotyl (Fig. 6O-Q).
GCT and CCT are transiently required for SAM and
vascular specification
The observation that gct and cct delay the expression of KAN1 and
KAN2 as well as provascular and cotyledon differentiation indicates
that these genes play a key role in temporal regulation of embryo
pattern formation. To more broadly characterize the function of GCT
and CCT in pattern formation, we examined the expression of
molecular markers for the SAM (pSTM::GUS) (Kirch et al., 2003),
RAM (pWOX5::GFP) (Xu et al., 2006) and vascular tissue (E2331)
in wild-type and mutant embryos. In wild type, the expression
of pSTM::GUS is first detected at the transition stage of
embryogenesis, just as cotyledon primordia emerge on the flanks of
the SAM (compare Fig. 7A and 7B). The size of the STM expression
domain then increases, reaching its maximum extent at the bent
cotyledon stage (Fig. 7C-E). In situ hybridization with an STM probe
demonstrated that the SAM of mature gct and cct embryos is
approximately 2.5 times larger than normal (Fig. 7E,I,M). Although
pSTM::GUS is expressed much later in mutant embryos than in their
wild-type siblings, in both genotypes pSTM::GUS expression is first
detected at the morphological heart stage (Fig. 7H,L). Thus, gct and
cct delay pSTM::GUS expression in absolute time, but not with
DEVELOPMENT
(right, Fig. 5G,H) display a disorganized cell division pattern.
This class probably represents a subsequent stage in the
development of the severely affected globular embryos illustrated
in Fig. 5D,E (right). At the wt late heart stage (Fig. 5I), the
majority of gct (left, Fig. 5J) and cct (left, Fig. 5K) embryos have
formed two small cotyledon primordia, and show increasingly
normal histological organization, such as the presence of
elongated vascular precursor cells. By contrast, a small number
of gct and cct embryos (right, Fig. 5J,K) continue to divide in a
highly disorganized fashion. Despite this, gct and cct embryos
have the same number of cells as wild-type embryos through the
early heart stage of development, demonstrating that the
developmental delay in these mutants is not a consequence of a
general reduction in growth rate (Fig. 5L). A significant difference
in cell number is only observed at the late heart stage, and this is
completely attributable to the onset of cotyledon development in
wild-type embryos at this stage.
At the bent cotyledon stage, the majority of gct and cct embryos
have elongated – albeit short – cotyledons, and an unusually large
SAM (Fig. 6B,G,D,I), whereas a small percentage display only
rudimentary cotyledons and completely lack a SAM (Fig. 6C,H,E,J).
The frequency of this latter class is similar to the frequency of
embryos that lack cotyledons at the late heart stage of development
(Fig. 5J,K), suggesting that this is the mature morphology of these
severely affected embryos. gct and cct also affect the organization
120
RESEARCH ARTICLE
Development 137 (1)
Fig. 8. gct and cct dissociate growth from temporal control of
early embryo patterning. A summary of the results presented in this
manuscript. During early embryogenesis, growth (i.e. cell division)
proceeds at the same rate in wild-type, gct and cct embryos (gray bars).
By contrast, radial patterning in gct and cct embryos is delayed for
several days compared with wild type (dashed black bar), after which it
resumes normally (solid black bar). gct and cct embryos express markers
for STM, PHB, FIL and WOX5 consistent with their stage of
morphogenesis (i.e. normally), whereas expression of markers for
KAN1, KAN2 and the vascular enhancer trap E2331 is delayed both
temporally, and with respect to the morphology of the embryo.
respect to developmental stage. This suggests that the delay in STM
expression is a consequence of delayed patterning. By contrast, gct
and cct do not affect the timing of pWOX5::GFP. This RAM marker
was expressed in all but the most phenotypically severe gct and cct
embryos at both the wild-type heart and bent cotyledon stages (see
Fig. S4 in the supplementary material).
The enhancer trap E2331 expresses GFP brightly in vascular
tissue starting at the early globular stage and throughout the rest of
embryonic development (Fig. 7N-Q), and is therefore an excellent
marker for vascular patterning. gct and cct embryos do not express
E2331 at the globular or heart stages (Fig. 7R,S,V,W). E2331
expression begins in the basal part of the hypocotyl of torpedo stage
gct and cct embryos (Fig. 7T,X), and by the bent cotyledon stage, it
is expressed throughout the vascular tissue of mutant embryos (Fig.
7U,Y); at this stage, the expression pattern of E2331 in mutant
embryos is essentially normal (Fig. 7Q). Thus, gct and cct delay the
expression of E2331 both in absolute time and relative to the
morphogenetic stage of the embryo, suggesting that they play a
direct role in vascular patterning.
DISCUSSION
Pattern formation is a result of temporally and spatially regulated
changes in gene expression. Although considerable attention has
been paid to the spatial component of this problem, the temporal
regulation of developmental patterning is an open question. Here we
show that growth and pattern formation can be dissociated during
embryogenesis in Arabidopsis, and demonstrate a role for
GCT/MED13 and CCT/MED12 in the temporal regulation of radial
polarity (summarized in Fig. 8).
GCT and CCT regulate the timing of genes
required for peripheral-abaxial identity
Peripheral-abaxial identity is specified by the KANADI family of
transcription factors. gct and cct mutations delay the onset of KAN1
and KAN2 expression, implying that GCT and CCT directly or
indirectly promote the expression of these genes early in
embryogenesis. Interestingly, the absence of KAN1 and KAN2
expression early in development is not accompanied by a change in
DEVELOPMENT
Fig. 7. Delayed expression of STM and the provascular enhancer
trap E2331 in gct and cct. (A-D)Expression of a pSTM::GUS
transcriptional fusion (Kirch et al., 2003) in wild-type globular (A),
transition (B), heart (C) and torpedo (D) stage embryos. (E)Detection of
STM mRNA by in situ hybridization in wild-type bent cotyledon stage
embryos. (F-M)pSTM::GUS expression is not seen in gct or cct embryos
at the transition (F,J) or heart (G,K) stages. Torpedo stage gct and cct
embryos initiate pSTM::GUS expression (H,L), and bent cotyledon stage
gct and cct embryos show an expanded STM expression domain (I,M).
(N-Q)Enhancer trap marker E2331 is expressed in vascular primorida of
globular (N), heart (O), torpedo (P) and bent cotyledon (Q) stage
embryos. (R-Y)gct and cct embryos at the globular (R,V) and heart
(S,W) stages do not express E2331. Beginning at the torpedo stage,
E2331 expression begins in lower vascular primordia of gct (T) and cct
(X) embryos. At the bent cotyledon stage, E2331 is expressed
throughout vascular primordia of gct (U) and cct (Y) embryos. Scale
bars: 20m.
RESEARCH ARTICLE
121
the expression of at least two other genes involved in the
specification of adaxial-abaxial polarity – the abaxial gene, FIL, and
the adaxial gene, PHB. This result provides an exception to the
antagonism that is generally observed between KAN and PHB gene
expression: typically, a decrease in KAN1 and KAN2 expression in
leaves is associated with an increase in the PHB expression domain
(Eshed et al., 2004). This antagonistic relationship has also been
observed in early embryogenesis (Grigg et al., 2009). One
explanation for the lack of an increased PHB expression domain in
gct and cct is that at the heart stage there are residual levels of KAN1
and KAN2 gene expression that cannot be detected by our reporter
constructs. Another possibility is that GCT and CCT are required to
mediate the antagonistic interactions between PHB and KAN. If this
is the case, further studies on the role of GCT and CCT may lead to
an understanding of the molecular nature of PHB-KAN antagonistic
interactions.
In light of the importance of the phytohormone auxin in
apicobasal and radial patterning of Arabidopsis embryos (Nawy et
al., 2008), it is interesting to note that both the abnormal orientation
of the first division of the apical cell in gct and cct and the resultant
double octant embryos are also observed in monopteros and
bodenlos auxin response mutants (Berleth and Jürgens, 1993;
Hamann et al., 1999). The phenotypic similarity of these four
mutants points to a possible role for GCT and CCT in promoting
auxin transcriptional responses in early embryogenesis. The
relationship between GCT, CCT and auxin is an exciting area for
future research.
apparently unrelated to developmental timing (Wang and Chen,
2004). This divergence in function between MED12/13 and CDK8
parallels studies in Drosophila, where, despite their physical
interaction, mutations in Med12 and Med13 condition different
phenotypes from Cdk8 mutants (Loncle et al., 2007).
GCT and CCT are required for temporal
coordination of growth and pattern formation
during embryonic development
Mature gct and cct embryos typically possess an enlarged SAM
and abnormally shaped cotyledons. The basis for the phenotype
became apparent when we examined the effect of these mutations
on the rate of cell division and the timing of various events in
embryo development. Whereas gct and cct had little or no effect
on the rate of cell division early in embryo development, they
delayed key morphogenetic events, including production of
provascular tissue, cotyledon initiation and the initiation of the
SAM. We suspect that this delay explains the effect of these
mutations on meristem size, because the effect does not persist
after germination, as is the case for mutations in meristem
maintenance genes such as WUS and the CLV loci (Schoof et al.,
2000). Temporal defects were also apparent at the level of gene
expression, and were of two types. Some genes – such as STM,
PHB and FIL – were expressed later than normal, but at the correct
morphological stage. Others – specifically KAN1, KAN2 and the
provascular marker E2331 – were delayed in time, with respect to
the morphology of the embryo, and also relative to genes with
which their expression is normally synchronized.
These results demonstrate the existence of a mechanism for
temporally coordinating events in plant embryogenesis, and indicate
that GCT and CCT are important components of this mechanism.
Interestingly, among the Arabidopsis Mediator genes that have been
functionally characterized, this role in temporal regulation of
development is apparently specific to GCT/MED13 and
CCT/MED12. For example, mutations in the Arabidopsis core
Mediator components SWP (MED14) and MED21 result in severely
dwarfed and lethal phenotypes (respectively) (Autran et al., 2002;
Dhawan et al., 2009). By contrast, loss-of-function mutations in
Arabidopsis HEN3/CDK8, a protein that in Drosophila physically
interacts with MED12 and MED13, have a subtle phenotype that is
Acknowledgements
J. Haxby isolated and initially characterized the E2023 and E2331 lines. We are
grateful to K. Gallagher for the use of a 20⫻ water immersion objective. M.
Heisler (pFIL::dsRed-N7), W. Werr (pSTM::GUS) and R. Heidstra (pWOX5::GFP)
kindly provided reporter constructs. L. Gutiérrez-Nava, M. Willmann, K. Earley,
C. Hunter, K.G. and D. Wagner provided helpful discussions. Thanks to D.
Grimanelli, W. Lukowitz, P. Jenik, G. Wu, M.W. and K.E. for comments on the
manuscript. This work was funded by an NIH NRSA fellowship to C.S.G.
(5F32GM069104-03), and by a DOE grant to R.S.P. and C.S.G. (DE-FG0299ER20328). Deposited in PMC for release after 12 months.
Transient requirement for Arabidopsis MED13 and
MED12 in embryo patterning
With rare exceptions (Carrera et al., 2008), yeast and animal Med13
and Med12 proteins act as transcriptional repressors (Samuelsen et
al., 2003; Ding et al., 2008; Knuesel et al., 2009). With this in mind,
one attractive hypothesis is that GCT and CCT regulate the maternalto-zygotic transition in early embryogenesis, perhaps by repressing
zygotic transcription during the window of early embryogenesis
when development is thought to be predominantly under maternal
control (Pagnussat et al., 2005; Grimanelli et al., 2005) (reviewed
by Baroux et al., 2008). In this scenario, the delayed pattern
formation and morphogenesis seen in early gct and cct embryos
would be attributed to a confusion in cell and tissue identity caused
by a heterochronic overlap of normally distinct maternal and zygotic
patterning programs. This overlap would be relieved after the first
few days of embryogenesis, when zygotic transcription normally
predominates, and would explain the delayed initiation of radial
patterning in gct and cct embryos. Regardless of the mechanism,
identification of the direct targets of GCT and CCT is a high priority
for future research, and should illuminate the role that these genes
play in temporal control of radial patterning in early embryogenesis.
Author contributions
C.S.G. designed and performed all experiments with the exception of in situ
hybridizations (M.Y.P.), and molecular and genetic characterization of the
E2023 line (M.R.S., R.P. and R.A.K.). C.S.G and R.S.P. analyzed data and wrote
the manuscript.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043174/-/DC1
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