RESEARCH ARTICLE 113

Development 137, 113-122 (2010) doi:10.1242/dev.043174 The MED12-MED13 module of 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 , GRAND CENTRAL (GCT)andCENTER 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 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.

KEY WORDS: Arabidopsis, Embryo, Heterochrony, MEDIATOR, MED12, MED13, Polarity, KANADI

INTRODUCTION (McConnell and Barton, 1998). Conversely, phb phv rev triple Plant embryos exhibit both apicobasal and central-peripheral (radial) mutants lack a SAM (Emery et al., 2003). Ectopic expression of polarity. The shoot and root apical meristems (SAM and RAM, KAN1 in the central domain of the embryo blocks the development respectively) form at opposite ends of the apicobasal axis and of the SAM, whereas kan1 kan2 kan4 triple mutant embryos have contain stem cell populations that give rise to all postembryonic outgrowths on the abaxial surface of cotyledons and the periphery tissue. Lateral organs such as cotyledons arise at the boundary of the of the hypocotyl that have characteristics of leaf primordia central and peripheral domains, and adopt the pattern present in the (Kerstetter et al., 2001; Izhaki and Bowman, 2007). radial axis of the embryo. Thus, genes that specify central identity The studies described above have begun to address the are also expressed on the adaxial (upper) side of developing developmental role of KAN genes in embryonic and postembryonic cotyledon primordia, whereas genes that specify peripheral identity development. However, the mechanism responsible for the initial are expressed on the abaxial (lower) side. This continuity supports establishment of radial polarity in embryogenesis remains a mystery. the conclusion that the adaxial surface of a lateral organ is a To identify factors in the polarity pathway that act upstream of KAN component of the central domain, whereas the abaxial side is a genes, we took advantage of an enhancer trap insertion in KAN2 that component of the peripheral domain (Kerstetter et al., 2001; expresses green fluorescent (GFP) in the peripheral-abaxial McConnell et al., 2001). domain of both embryonic and postembryonic organs. A screen for Genes involved in the specification of radial polarity were initially mutations that affect the expression of this reporter produced alleles identified in screens for mutations that affect the adaxial-abaxial of two genes, GRAND CENTRAL (GCT) and CENTER CITY (CCT), polarity of leaves and floral organs. In Arabidopsis, these screens which we found encode the Arabidopsis homologs of MED13 and have produced mutations in three families of transcription factors. MED12. Members of the class III HD-ZIP family of transcription factors In yeasts and animals, Med13 and Med12 act as transcriptional promote adaxial identity (McConnell et al., 2001; Emery et al., repressors by inhibiting core Mediator, a multisubunit complex that 2003), whereas KANADI (KAN) and YABBY (YAB) transcription allows transcription factors bound at upstream enhancer elements to factor families promote peripheral and abaxial identity (Kerstetter activate RNA polymerase II. Med13 and Med12 have identical et al., 2001; Eshed et al., 2001; Siegfried et al., 1999). Evidence that mutant phenotypes in each organism (Samuelsen et al., 2003; Yoda these genes are also important for embryonic development is et al., 2005; Janody et al., 2003), indicating that they have similar provided by the phenotype of gain-of-function alleles, and the biological functions. In humans, MED13 negatively regulates phenotype of embryos lacking more than one member of these transcription by causing an allosteric change in core Mediator that families. For example, mutations that cause the HD-ZIP III gene prevents its association with RNA polymerase II (Knuesel et al., PHB to be ectopically expressed in the peripheral-abaxial domain 2009), whereas MED12 recruits the histone methyltransferase G9a produce SAMs (central structures) on the abaxial side of leaves 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 Cyclin- 1Department of Biological Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. 2Department of Plant Biology and Pathology, The Waksman Institute, dependent kinase 8 (Cdk8) and Cyclin C (CycC) to form a complex Rutgers University, Piscataway, NJ 08854, USA. known as the Cdk8 module of Mediator (Samuelsen et al., 2003; Loncle et al., 2007). However, the observation that in Drosophila *Author for correspondence ([email protected]) Cdk8 and CycC do not have the same mutant phenotype as Med13

Accepted 9 October 2009 (skd – FlyBase) and Med12 (kto – FlyBase), implies that these DEVELOPMENT 114 RESEARCH ARTICLE Development 137 (1) components are functionally distinct (Loncle et al., 2007). In both 2002) were obtained from the Arabidopsis Stock Center (ABRC) at Drosophila and Caenorhabditis elegans, Med13 and Med12 specify www.arabidopsis.org. Seed for gct-2, cct-1, E2023, E2331 and gPHB-GUS cell identity by regulating downstream targets of the Wnt signaling have been deposited in the Arabidopsis Stock Center (ABRC). pathway (Janody et al., 2003; Yoda et al., 2005; Carrera et al., 2008). E2023 seeds were mutagenized as follows: 100 mg of seed was In particular, the Drosophila Med13 and Med12 genes skuld and suspended in 25 ml of water with 3 l Tween-20 for 15 minutes, rinsed and kohtalo control the cell affinities that maintain boundaries between 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 anteroposterior and dorsoventral compartment boundaries of phosphate buffer (pH 7) for 9 hours on a rotator at room temperature, and the wing disc (Janody et al., 2003; Loncle et al., 2007). The subsequently rinsed 10 times with 25 ml of water. Embryos from three developmentally specific phenotypes of mutations in Med13 and consecutive siliques on the main inflorescence of 2500 M1 plants were Med12 in Drosophila and C. elegans are consistent with a examined for changes in E2023 expression and embryo morphology. The microarray analysis of these genes in yeast, which indicates that they main inflorescence of M1 plants was consistently found to have two or three regulate a relatively small number of genes (Samuelsen et al., 2003). sectors, and thus our screen comprised 5000-7500 individual mutagenized This is in contrast to core Mediator, which is required for nearly all sectors. Mutant lines were backcrossed three times before analysis. transcription (Kornberg, 2005). Molecular biology The core Mediator complex was recently purified from RNA in situ hybridization was performed using a protocol obtained from Arabidopsis suspension culture cells (Bäckström et al., 2007). The Jeff Long (www.its.caltech.edu/~plantlab/protocols/insitu.pdf), with the purified complex contained most of the components present in the following minor modifications. The probe was hybridized to slides at a core complex in other organisms, but was missing proteins in the temperature of 60-65°C overnight. The blocking and antibody dilution Cdk8 module, consistent with the limited and transient interactions solutions were produced using maleic acid instead of Tris-HCl. For the STM, observed between these proteins and core Mediator in other systems GCT and CCT probes, partial cDNA sequences were amplified and (Andrau et al., 2006). The phenotype of mutations in two transcribed using the following primers: STM-T7, 5Ј-CAAGCTTA - ATACGACTCACTATAGGGAAAGGATTGCCCAAGACAT-3Ј; STM- components of the core complex (PFT1/MED25 and SWP/MED14) Ј has been described in Arabidopsis. Mutations of PFT1/MED25 SP6, 5 -CTCTAGATTTAGGTGACACTATAGGATCCATC ATAA C G - AAATCGT-3Ј; GCT-SP6, 5Ј-ATTTAGGTGACACTATA GAAGC CC - delay flowering under suboptimal light conditions (Cerdán and CAAATTAGTTCTACAAGTGG-3Ј; GCT-T7, 5Ј-TAATA CGAC - Chory, 2003; Bäckström et al., 2007), whereas mutations of TCACTAT AGGGAGACCAGGTCCAGAACCTCTGC-3Ј; CCT-SP6, SWP/MED14 cause a premature arrest in cell proliferation during 5Ј-ATTTAG GTGACACTATAGAAGAAGCTTGTATCTGTATT AGATT - vegetative growth, resulting in extreme dwarfing (Autran et al., GC-3Ј; CCT-T7, 5Ј-TAATACGACTCACTATAGGGAGACCTTC CT - 2002). By contrast, mutations in the Arabidopsis Cdk8 homolog CAATCACTTCC CC-3 Ј. HEN3 have a mild reduction in cell expansion in leaves, and only The gPHB-GUS translational fusion construct was made by cloning show more severe phenotypes in combination with mutations that 10443 bp upstream of the PHB stop codon in front of the GUS+ gene in the affect pre-mRNA processing (Wang and Chen, 2004). pCAMBIA3300 vector, with an NAAIRS linker replacing the PHB stop codon. This DNA fragment contains all sequences from the previous gene Here we show that MED13 and MED12 regulate developmental Ј patterning in Arabidopsis. We found that MED13, corresponding to 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 the GCT gene, and MED12, corresponding to the CCT gene, are construct show mild adaxialized phenotypes, indicating that the PHB-GUS absolutely required for KAN expression during early embryogenesis, fusion protein is biologically active. and promote KAN expression during postembryonic development as well. Analysis of markers for the SAM and vascular tissue Microscopy and histology showed that gct and cct mutations have a unique effect on embryo The E2023 genetic screen was conducted using a Leica MZFLIII patterning: the initiation of a number of patterning genes is delayed fluorescence microscope equipped with a long pass GFP filter. To detect for several days, after which they are expressed in an almost normal GUS activity, embryos were stained at 37°C for 1h (pKAN::GUS, pSTM::GUS) or overnight (gPHB-GUS) according to Donnelly et al. manner. The expression pattern and proposed biochemical function (Donnelly et al., 1999). Embryos were cleared in Hoyer’s solution (bent of GCT and CCT is consistent with a role for these genes in cotyledon stage embryos) or mounted in 5% glycerol (globular to torpedo transiently repressing transcription of genes that would otherwise stage) and examined with DIC optics. Confocal images were obtained using interfere with radial pattern formation. Our results reveal a role for a Leica TCS SL confocal microscope with a 20ϫ water immersion lens. MED12 and MED13 in the regulation of developmental timing, and Embryos were removed from ovules using DuMont #5 forceps and mounted uncover a novel temporal component of pattern formation in in 1ϫMS salts plus 500 mM glucose. Globular and heart stage embryos Arabidopsis embryogenesis. 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 MATERIALS AND METHODS water, and manually sketched using an Olympus BX40 microscope Genetic stocks and growth conditions equipped with a U-DA light tube and mirror. All seed stocks are in the Columbia ecotype. Enhancer trap lines E2023 and E2331 were generated using the pBIN 35S mGAL4-VP16+UAS mGFP5- ER vector, containing a minimal promoter driving ER localized mGFP5 RESULTS (Haseloff, 1999). E2023 contains at least two copies of the GAL4- A genetic screen to identify upstream regulators UAS::GFP enhancer trap in inverted orientation in the second intron of the of peripheral-abaxial polarity KAN2 gene (at bp 2889 of the genomic sequence relative to start codon), as The KAN family of transcription factors specify peripheral-abaxial well as a second linked T-DNA insertion between genes At1g29880 and identity beginning in early embryogenesis (Kerstetter et al., 2001; At1g29890. The enhancer sequences driving E2331 expression have not been defined. The kan1-12 allele (Kerstetter et al., 2001) and the kan2-4 Eshed et al., 2004). To identify genes that act upstream of KAN in allele (SALK_095643) (Wu et al., 2008) were used for double mutant the embryo polarity pathway, we took advantage of the enhancer analysis. Plants were grown under 16 hours fluorescent illumination (100 trap line E2023, which contains a GAL4-UAS::GFP insertion E/m2; Sylvania VHO) at 22°C in Farfard #2 soil. SALK T-DNA insertion (Haseloff, 1999) that disrupts the KAN2 gene (see Materials and

lines (Alonso et al., 2003) and SAIL T-DNA insertion lines (Sessions et al., methods). This line expresses GFP strongly and constitutively DEVELOPMENT Temporal control of embryo patterning RESEARCH ARTICLE 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: 2ϫ 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. within the peripheral-abaxial domain of embryos, the abaxial significant phenotypic differences were noted between the various vasculature of leaves and the abaxial domain of carpels (Fig. 1A-F), alleles of gct; gct-2 and cct-1 (hereafter referred to as gct and cct) an expression pattern that is essentially identical to that of KAN2 were used for all of the experiments presented here. Homozygous (Emery et al., 2003; Eshed et al., 2004). It has no obvious plants produce very few seeds, and thus gct and cct embryos were morphological defect beyond the production of a small longitudinal obtained from heterozygous plants, allowing us to stage the ridge of tissue below the first bract, and an occasional flower with development of mutant embryos by observing wild-type embryos five petals (data not shown). segregating in the same silique. Thus, for all analyses in this Examination of approximately 6000 mutagenized M1 sectors manuscript, the developmental stage of mutant embryos was (see Materials and methods) yielded more than 50 mutant lines inferred from wild-type siblings of the same chronological age. with a heritable effect on E2023 expression. Five of these lines GCT and CCT also regulate the expression of E2023 during segregated embryos with a nearly identical recessive mutant postembryonic development, and affect the timing of a variety of phenotype, consisting of a decrease in the intensity of E2023 developmental transitions including the embryo-to-seedling expression (Fig. 1G-J,M-P), and an increase in size of the SAM transition, vegetative phase change and flowering time. This (see below). Complementation analysis revealed that these paper focuses primarily on their role in embryogenesis. mutations represented two genes, which were named GRAND GCT and CCT are absolutely required for E2023 expression at the CENTRAL (GCT) (four alleles) and CENTER CITY (CCT) (one heart stage of embryogenesis, and promote the correct level of allele), to reflect the increased size of the SAM and the decrease expression later in embryo development (Fig. 1G-J,M-P). E2023 is in expression of the peripheral-abaxial enhancer trap E2023. No 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-week- old 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 116 RESEARCH ARTICLE 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. 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 Fig. 3. GCT and CCT promote KAN1 expression in early mutants resemble gct, but differ from gct in having ectopic growths embryogenesis. (A-D)gPHB-GUS is expressed in the SAM and vascular on both the cotyledons and hypocotyl (arrowheads, Fig. 2D). These primodia of wild-type transition stage embryos (A), on the adaxial side of ectopic growths have previously been observed only in kan1 kan2 cotyledons and in vascular primordia of wild-type heart stage embryos (B), in vascular primordia and the adaxial epidermis of cotyledons in wild- kan4 triple mutants (Izhaki and Bowman, 2007). type torpedo stage embryos (C) and in the SAM of bent cotyledon gct also enhanced kan1 postembryonic phenotypes. Whereas embryos (D). A similar pattern is observed in gct (E-H) and cct (I-L) the leaves of kan1 and gct are held horizontally (Fig. 2E-G), the embryos, with the exception that gPHB-GUS is not expressed in the leaves of gct kan1 double mutants stand upright, curl upwards and adaxial epidermis of cotyledons. (M-O)A pFIL::dsRed-N7 transcriptional are irregularly expanded (Fig. 2H). This effect on leaf fusion is expressed on the abaxial face of cotyledons in torpedo stage morphology is associated with a change in polarity of mesophyll wild-type (M) gct (N) and cct (O) embryos. (P-R)Expression of a cells. Adaxial (palisade) mesophyll cells are typically round and pKAN1::GUS transcriptional fusion in the peripheral domain of wild-type densely packed, whereas abaxial (spongy) mesophyll cells are embryos at the heart (P), torpedo (Q) and bent cotyledon stages (R). No irregular in shape and are separated by large air spaces (Fig. 2I). expression of pKAN1::GUS in heart stage gct (S) and cct (V) embryos, kan1 (Fig. 2J), and to a lesser extent gct (Fig. 2K), cause spongy initiation of expression in torpedo stage gct (T) and cct (W) embryos, and reduced but spatially normal expression in wild-type bent cotyledon stage mesophyll cells to be less convoluted and more highly packed gct (U) and cct (X) embryos. Scale bar: 20m. 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 GCT and CCT regulate KAN expression intermediate between wild-type adaxial and abaxial mesophyll independently of PHB cells. Although kan1 and gct do not have a major effect on carpel To further characterize the role of GCT and CCT in the specification development (Fig. 2M-O), gct kan1 flowers (Fig. 2P) have an of adaxial-abaxial polarity, we examined the effect of gct and cct enlarged septum with increased stigmatic tissue, less valve tissue mutations on expression of a representative member of the KAN, YAB and abaxial projections of carpel and papillary tissue, and are and class III HD-ZIP gene families. The pKAN1::GUS construct (Wu essentially identical to the flowers of kan1 kan2/+ plants (Pekker et al., 2008) expresses GUS in the peripheral domain of the hypocotyl et al., 2005). These results are consistent with the decreased throughout embryogenesis (Fig. 3P-R). gct and cct delayed the expression of E2023 in gct, and suggest that GCT contributes to expression of pKAN1::GUS until the torpedo stage of development the specification of peripheral-abaxial identity by promoting the (Fig. 3S-T,V-W) and slightly reduced its level of expression, but by

expression of KAN2. the bent cotyledon stage had no obvious effect on its spatial expression DEVELOPMENT Temporal control of embryo patterning RESEARCH ARTICLE 117

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.

pattern (Fig. 3R,U,X). Thus, gct and cct have the same effect on the phenotype of gct-2 (Fig. 4A). CCT is located at the top of expression of KAN1 as they have on the expression of KAN2 (Fig. 1G- 4 and corresponds to At4g00450; cct-1 is a missense J,M-P). By contrast, gct and cct did not affect the expression of mutation in At4g00450, and two T-DNA insertions in At4g00450 pFIL::dsRed-N7, a reporter for the abaxially expressed gene (SALK_108241 and SALK_124276) have a phenotype identical to YABBY1/FILAMENTOUS FLOWER (FIL) (Heisler et al., 2005) (Fig. cct-1 and failed to complement this point mutation (Fig. 4B). 3M-O; see Fig. S1 in the supplementary material). GCT and CCT are single copy genes in Arabidopsis. The predicted Class III HD-ZIP genes interact antagonistically with KAN genes GCT protein has 1921 amino acids (AA), and contains a serine-rich (Eshed et al., 2004). To determine if the decrease in KAN1 and KAN2 region between AA 407 and 426. gct-3 changes the first serine of this expression in gct and cct embryos is the result of an increase in HD- domain to an asparagine, suggesting that this domain is functionally ZIPIII expression, we examined their effect on expression of the important. AA 1088-1179 of the predicted GCT protein are 42% HD-ZIPIII gene PHB. Because PHB is regulated post- identical to the MED13 pfam06333 domain (Fig. 4A). The entire transcriptionally by miR165/miR166, this experiment was predicted GCT protein is 16% identical and 19% similar to human conducted using a reporter in which GUS was fused to the C- MED13, and regions of identity and similarity are distributed terminal end of a PHB genomic clone (gPHB-GUS). This construct throughout the protein (see Fig. S2 in the supplementary material). has an expression pattern identical to that reported for PHB mRNA The predicted CCT protein is 2144 AA long; AA 69-127 are 37% (McConnell et al., 2001) (Fig. 3A-D). Interestingly, although gct and identical to the MED12 pfam09497 domain (Fig. 4B). The entire cct reduced gPHB-GUS expression on the adaxial side of predicted CCT protein is 12% identical and 21% similar to human cotyledons, the timing and overall GUS pattern in these mutants was MED12 (NP_056150), and regions of identity and similarity are essentially normal. Thus, the decrease in KAN expression in gct and distributed throughout the protein (see Fig. S3 in the supplementary cct is not attributable to an increase in the size of the PHB expression material). These results suggest that the MED12/MED13 regulatory domain, or to an increase in the level of PHB expression. module of Mediator is conserved in plants.

GCT and CCT encode MED13 and MED12, predicted GCT and CCT are dynamically expressed during transcriptional repressors conserved in all embryo development eukaryotes The expression of GCT and CCT during embryogenesis was The molecular identities of GCT and CCT were determined using a characterized by RNA in situ hybridization. GCT mRNA was map-based approach (Lukowitz et al., 2000). GCT is located on the detected in both the apical and basal cell at the one-cell embryo stage lower arm of chromosome 1 and corresponds to At1g55325; all four (Fig. 4C), whereas CCT was first detected at the dermatogen stage EMS-induced gct alleles contained mutations in At1g55325, and a of embryogenesis (Fig. 4L). Both genes are expressed in all cells of T-DNA insertion in At1g55325 (SAIL_1169_H11) has a phenotype the embryo proper and suspensor through the globular stage of

identical to these EMS alleles and failed to complement the mutant embryogenesis (Fig. 4C-F,L-M), but their expression begins to DEVELOPMENT 118 RESEARCH ARTICLE Development 137 (1)

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.

decrease in the periphery of the hypocotyl and on the abaxial side of In both gct and cct the first division of the apical cell is transverse cotyledons starting at the heart stage (Fig. 4G,N). This trend to the long axis of the embryo, instead of parallel as in wild type continues through the torpedo stage (Fig. 4H,O), and by the bent (arrows, Fig. 5A,B); furthermore, cells of the embryo proper cotyledon stage GCT and CCT mRNAs are restricted to the vascular (brackets) are abnormally elongated, and in this respect resemble tissue and SAM and RAM (Fig. 4I-K,P-R). This pattern is consistent suspensor cells (marked by the dashed line). gct (Fig. 5D) and cct with the temporal effect of gct and cct mutations on the expression (Fig. 5E) globular embryos are more elongated than wild type (Fig. of E2023 and pKAN1::GUS (Figs 1 and 3), and suggests that these 5C), and have more suspensor cells. The more severely affected genes are important in the peripheral domain of the embryo only embryos also lack the lens-shaped cell produced by asymmetric early in embryogenesis. Postembryonically, public microarray division of the uppermost cell of the suspensor (asterisks in Fig. expression data indicate that GCT and CCT are expressed in most 5D,E), and have one or more extra tiers of cells between the tissue types, with highest expression in the shoot apex (Winter et al., suspensor and the lower tier of the embryo (‘x’ in Fig. 5D,E). These 2007). extra tiers probably result from the abnormal first division plane of the apical cell shown in Fig. 5A,B. Although the percentage of gct and cct uncouple cell division, pattern phenotypically mutant gct and cct embryos segregating in siliques formation, and morphogenesis of heterozygous plants is quite low at the octant, dermatogen and Our analysis of GCT and CCT expression demonstrated that their globular stages (approximately 5%), these phenotypes were never mRNAs are present in wild-type embryos before the initiation of observed in octant (n227), dermatogen (n215) or globular stage KAN expression. To explore additional roles for GCT and CCT in (n344) embryos from wild-type plants. early embryogenesis, the histological phenotype of wild-type, gct In wild-type embryogenesis, the onset of the heart stage of and cct embryos was characterized from the earliest stages of development is marked by the initiation of cotyledon primordia development. We found that pattern formation and morphogenesis on the upper flanks of the embryo, and the appearance of are delayed in gct and cct embryos compared with wild type (Fig. elongated vascular cells (Fig. 5F). gct and cct delay both of these

5A-K). patterning events (Fig. 5G,H). The most strongly affected mutants DEVELOPMENT 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 wild- type (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.

(right, Fig. 5G,H) display a disorganized cell division pattern. of the RAM and root cap, but to a lesser extent than the SAM (Fig. This class probably represents a subsequent stage in the 6K-N). gct and cct increase the size of hypocotyl cells, but do not development of the severely affected globular embryos illustrated change the number of tissue layers in the hypocotyl (Fig. 6O-Q). 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 GCT and CCT are transiently required for SAM and formed two small cotyledon primordia, and show increasingly vascular specification normal histological organization, such as the presence of The observation that gct and cct delay the expression of KAN1 and elongated vascular precursor cells. By contrast, a small number KAN2 as well as provascular and cotyledon differentiation indicates of gct and cct embryos (right, Fig. 5J,K) continue to divide in a that these genes play a key role in temporal regulation of embryo highly disorganized fashion. Despite this, gct and cct embryos pattern formation. To more broadly characterize the function of GCT have the same number of cells as wild-type embryos through the and CCT in pattern formation, we examined the expression of early heart stage of development, demonstrating that the molecular markers for the SAM (pSTM::GUS) (Kirch et al., 2003), developmental delay in these mutants is not a consequence of a RAM (pWOX5::GFP) (Xu et al., 2006) and vascular tissue (E2331) general reduction in growth rate (Fig. 5L). A significant difference in wild-type and mutant embryos. In wild type, the expression in cell number is only observed at the late heart stage, and this is of pSTM::GUS is first detected at the transition stage of completely attributable to the onset of cotyledon development in embryogenesis, just as cotyledon primordia emerge on the flanks of wild-type embryos at this stage. the SAM (compare Fig. 7A and 7B). The size of the STM expression At the bent cotyledon stage, the majority of gct and cct embryos domain then increases, reaching its maximum extent at the bent have elongated – albeit short – cotyledons, and an unusually large cotyledon stage (Fig. 7C-E). In situ hybridization with an STM probe SAM (Fig. 6B,G,D,I), whereas a small percentage display only demonstrated that the SAM of mature gct and cct embryos is rudimentary cotyledons and completely lack a SAM (Fig. 6C,H,E,J). approximately 2.5 times larger than normal (Fig. 7E,I,M). Although The frequency of this latter class is similar to the frequency of pSTM::GUS is expressed much later in mutant embryos than in their embryos that lack cotyledons at the late heart stage of development wild-type siblings, in both genotypes pSTM::GUS expression is first (Fig. 5J,K), suggesting that this is the mature morphology of these detected at the morphological heart stage (Fig. 7H,L). Thus, gct and

severely affected embryos. gct and cct also affect the organization cct delay pSTM::GUS expression in absolute time, but not with DEVELOPMENT 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.

Fig. 7. Delayed expression of STM and the provascular enhancer E2331 at the globular or heart stages (Fig. 7R,S,V,W). E2331 trap E2331 in gct and cct. (A-D)Expression of a pSTM::GUS expression begins in the basal part of the hypocotyl of torpedo stage transcriptional fusion (Kirch et al., 2003) in wild-type globular (A), gct and cct embryos (Fig. 7T,X), and by the bent cotyledon stage, it transition (B), heart (C) and torpedo (D) stage embryos. (E)Detection of is expressed throughout the vascular tissue of mutant embryos (Fig. STM mRNA by in situ hybridization in wild-type bent cotyledon stage 7U,Y); at this stage, the expression pattern of E2331 in mutant 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 is essentially normal (Fig. 7Q). Thus, gct and cct delay the embryos initiate pSTM::GUS expression (H,L), and bent cotyledon stage expression of E2331 both in absolute time and relative to the gct and cct embryos show an expanded STM expression domain (I,M). morphogenetic stage of the embryo, suggesting that they play a (N-Q)Enhancer trap marker E2331 is expressed in vascular primorida of direct role in vascular patterning. 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 DISCUSSION (S,W) stages do not express E2331. Beginning at the torpedo stage, Pattern formation is a result of temporally and spatially regulated E2331 expression begins in lower vascular primordia of gct (T) and cct changes in gene expression. Although considerable attention has (X) embryos. At the bent cotyledon stage, E2331 is expressed been paid to the spatial component of this problem, the temporal throughout vascular primordia of gct (U) and cct (Y) embryos. Scale regulation of developmental patterning is an open question. Here we bars: 20m. 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 respect to developmental stage. This suggests that the delay in STM polarity (summarized in Fig. 8). expression is a consequence of delayed patterning. By contrast, gct and cct do not affect the timing of pWOX5::GFP. This RAM marker GCT and CCT regulate the timing of genes was expressed in all but the most phenotypically severe gct and cct required for peripheral-abaxial identity embryos at both the wild-type heart and bent cotyledon stages (see Peripheral-abaxial identity is specified by the KANADI family of Fig. S4 in the supplementary material). transcription factors. gct and cct mutations delay the onset of KAN1 The enhancer trap E2331 expresses GFP brightly in vascular and KAN2 expression, implying that GCT and CCT directly or tissue starting at the early globular stage and throughout the rest of indirectly promote the expression of these genes early in embryonic development (Fig. 7N-Q), and is therefore an excellent embryogenesis. Interestingly, the absence of KAN1 and KAN2

marker for vascular patterning. gct and cct embryos do not express expression early in development is not accompanied by a change in DEVELOPMENT Temporal control of embryo patterning RESEARCH ARTICLE 121 the expression of at least two other genes involved in the apparently unrelated to developmental timing (Wang and Chen, specification of adaxial-abaxial polarity – the abaxial gene, FIL, and 2004). This divergence in function between MED12/13 and CDK8 the adaxial gene, PHB. This result provides an exception to the parallels studies in Drosophila, where, despite their physical antagonism that is generally observed between KAN and PHB gene interaction, mutations in Med12 and Med13 condition different expression: typically, a decrease in KAN1 and KAN2 expression in phenotypes from Cdk8 mutants (Loncle et al., 2007). leaves is associated with an increase in the PHB expression domain (Eshed et al., 2004). This antagonistic relationship has also been Transient requirement for Arabidopsis MED13 and observed in early embryogenesis (Grigg et al., 2009). One MED12 in embryo patterning explanation for the lack of an increased PHB expression domain in With rare exceptions (Carrera et al., 2008), yeast and animal Med13 gct and cct is that at the heart stage there are residual levels of KAN1 and Med12 proteins act as transcriptional repressors (Samuelsen et and KAN2 gene expression that cannot be detected by our reporter al., 2003; Ding et al., 2008; Knuesel et al., 2009). With this in mind, constructs. Another possibility is that GCT and CCT are required to one attractive hypothesis is that GCT and CCT regulate the maternal- mediate the antagonistic interactions between PHB and KAN. If this to-zygotic transition in early embryogenesis, perhaps by repressing is the case, further studies on the role of GCT and CCT may lead to zygotic transcription during the window of early embryogenesis an understanding of the molecular nature of PHB-KAN antagonistic when development is thought to be predominantly under maternal interactions. control (Pagnussat et al., 2005; Grimanelli et al., 2005) (reviewed In light of the importance of the phytohormone auxin in by Baroux et al., 2008). In this scenario, the delayed pattern apicobasal and radial patterning of Arabidopsis embryos (Nawy et formation and morphogenesis seen in early gct and cct embryos al., 2008), it is interesting to note that both the abnormal orientation would be attributed to a confusion in cell and tissue identity caused of the first division of the apical cell in gct and cct and the resultant by a heterochronic overlap of normally distinct maternal and zygotic double octant embryos are also observed in monopteros and patterning programs. This overlap would be relieved after the first bodenlos auxin response mutants (Berleth and Jürgens, 1993; few days of embryogenesis, when zygotic transcription normally Hamann et al., 1999). The phenotypic similarity of these four predominates, and would explain the delayed initiation of radial mutants points to a possible role for GCT and CCT in promoting patterning in gct and cct embryos. Regardless of the mechanism, auxin transcriptional responses in early embryogenesis. The identification of the direct targets of GCT and CCT is a high priority relationship between GCT, CCT and auxin is an exciting area for for future research, and should illuminate the role that these genes future research. play in temporal control of radial patterning in early embryogenesis.

GCT and CCT are required for temporal Acknowledgements J. Haxby isolated and initially characterized the E2023 and E2331 lines. We are coordination of growth and pattern formation grateful to K. Gallagher for the use of a 20ϫ water immersion objective. M. during embryonic development Heisler (pFIL::dsRed-N7), W. Werr (pSTM::GUS) and R. Heidstra (pWOX5::GFP) Mature gct and cct embryos typically possess an enlarged SAM kindly provided reporter constructs. L. Gutiérrez-Nava, M. Willmann, K. Earley, and abnormally shaped cotyledons. The basis for the phenotype 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 became apparent when we examined the effect of these mutations manuscript. This work was funded by an NIH NRSA fellowship to C.S.G. on the rate of cell division and the timing of various events in (5F32GM069104-03), and by a DOE grant to R.S.P. and C.S.G. (DE-FG02- embryo development. Whereas gct and cct had little or no effect 99ER20328). Deposited in PMC for release after 12 months. on the rate of cell division early in embryo development, they delayed key morphogenetic events, including production of Author contributions C.S.G. designed and performed all experiments with the exception of in situ provascular tissue, cotyledon initiation and the initiation of the hybridizations (M.Y.P.), and molecular and genetic characterization of the SAM. We suspect that this delay explains the effect of these E2023 line (M.R.S., R.P. and R.A.K.). C.S.G and R.S.P. analyzed data and wrote mutations on meristem size, because the effect does not persist the manuscript. after germination, as is the case for mutations in meristem Supplementary material maintenance genes such as WUS and the CLV loci (Schoof et al., Supplementary material for this article is available at 2000). Temporal defects were also apparent at the level of gene http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043174/-/DC1 expression, and were of two types. Some genes – such as STM, PHB and FIL – were expressed later than normal, but at the correct References Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., morphological stage. Others – specifically KAN1, KAN2 and the Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R. et al. (2003). provascular marker E2331 – were delayed in time, with respect to Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, the morphology of the embryo, and also relative to genes with 653-657. Andrau, J.-C., van de Pasch, L., Lijnzaad, P., Bijma, T., Koerkamp, M. G., van which their expression is normally synchronized. de Peppel, J., Werner, M. and Holstege, F. C. P. (2006). Genome-wide These results demonstrate the existence of a mechanism for location of the coactivator Mediator: binding without activation and transient temporally coordinating events in plant embryogenesis, and indicate CDK8 interaction on DNA. Mol. Cell 22, 179-192. that GCT and CCT are important components of this mechanism. Autran, D., Jonak, C., Belcram, K., Beemster, T. S., Kronenberger, J., Granjean, O., Inzé, D. and Traas, J. (2002). Cell numbers and leaf Interestingly, among the Arabidopsis Mediator genes that have been development in Arabidopsis: a functional analysis of the STRUWWELPETER gene. functionally characterized, this role in temporal regulation of EMBO J. 21, 6036-6049. development is apparently specific to GCT/MED13 and Bäckström, S., Elfving, N., Nilsson, R., Wingsle, G. and Bjöklund, S. (2007). Purification of a plant Mediator from Arabidopsis thaliana identifies PFT1 as the CCT/MED12. For example, mutations in the Arabidopsis core Med25 subunit. Mol. Cell 26, 717-729. Mediator components SWP (MED14) and MED21 result in severely Baroux, C., Autran, D., Gillmor, C. S., Grimanelli, D. and Grossniklaus, U. dwarfed and lethal phenotypes (respectively) (Autran et al., 2002; (2008). The maternal to zygotic transition in animals and plants. Cold Spring Dhawan et al., 2009). By contrast, loss-of-function mutations in Harb. Symp. Quant. Biol. 73, 89-100. Berleth, T. and Jürgens, G. (1993). The role of the monopteros gene in Arabidopsis HEN3/CDK8, a protein that in Drosophila physically organizing the basal body region of the Arabidopsis embryo. Development 118,

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