Abstract

The phytohormone auxin is a key regulator of organogenesis in plants and is distributed asymmetrically via polar transport. However, the precise mechanisms underlying auxin-mediated organogenesis remain elusive. Here, we have analyzed the macchi-bou 2 (mab2) mutant identified in a pinoid (pid) enhancer mutant screen. Seedlings homozygous for either mab2 or pid showed only mild phenotypic effects on cotyledon positions and/or numbers. In contrast, mab2 pid double mutant seedlings completely lacked cotyledons, indicating a synergistic interaction. We found that mab2 homozygous embryos had defective patterns of cell division and showed aberrant cotyledon organogenesis. Further analysis revealed that the mab2 mutation affected auxin response but not auxin transport in the embryos, suggesting the involvement of MAB2 in auxin response during embryogenesis. MAB2 encodes an Arabidopsis ortholog of MED13, a putative regulatory module component of the Mediator complex. Mediator is a multicomponent complex that is evolutionarily conserved in eukaryotes and its regulatory module associates with Mediator to control the interaction of Mediator and RNA polymerase II. MAB2 interacts with a regulatory module component in yeast cells. Taken together, our data suggest that MAB2 plays a crucial role in embryo patterning and cotyledon organogenesis, possibly through modulating expression of specific genes such as auxin-responsive genes.

Introduction

In higher plants, aerial organs such as leaves and flowers are formed from the shoot apical meristem (SAM) in a highly ordered arrangement. Although this patterning implies a requirement for regulation through spatially and temporally coordinated programs, the precise mechanism remains elusive. The cotyledon is the first aerial organ to differentiate, and its initiation is inherent in the embryonic patterning program. In dicots, cotyledon initiation demarcates the change from radial to bilateral symmetry during embryogenesis, and the two initiation sites of the cotyledon primordia are defined symmetrically on either side of the SAM from the globular stage onward (Bowman and Floyd 2008). This establishment of bilateral symmetry at the apex of the globular embryo is correlated with changes in auxin distribution that are mediated by the auxin efflux carriers PIN-FORMED (PIN) family proteins (Friml et al. 2003, Blilou et al. 2005, Vieten et al. 2005). At least four PIN genes, PIN1, PIN3, PIN4 and PIN7, are differentially expressed during embryogenesis. Single mutants for each gene display only mild and infrequent early embryonic defects as a consequence of their redundant role in embryogenesis; however, severe embryonic defects including lack of an apical–basal body axis are observed in embryos carrying multiple mutations (Friml et al. 2003, Blilou et al. 2005).

Mutation of the genes that regulate the polar localization of PIN proteins can affect both local auxin distribution and embryo patterning. The guanine nucleotide exchange factor on ADP-ribosylation factor GTPases, GNOM, controls the recycling of PIN proteins from the endosome to the plasma membrane, and embryos with a mutation of this factor display severe defects in apical–basal body axis formation similar to multiple pin mutants (Mayer et al. 1993, Shevell et al. 1994, Busch et al. 1996, Geldner et al. 2003). The serine-threonine kinase protein PINOID (PID) is notable for directly regulating the polar localization of PIN proteins. Loss of PID activity leads to an apical to basal shift in PIN1 localization at the inflorescence apex and embryonic cotyledons (Benjamins et al. 2001, Friml et al. 2004, Treml et al. 2005). Conversely, gain of PID function causes a basal to apical shift in PIN1, PIN2 and PIN4 localization in seedling roots and developing embryos, leading to the loss of asymmetrical auxin distribution (Friml et al. 2004, Treml et al. 2005).

In the developing embryo, the directional flow of auxin mediated by PIN1 generates auxin maxima at the periphery. These auxin maxima induce activation of specific AUXIN RESPONSE FACTOR (ARF) genes such as MONOPTEROS (MP)/ARF5 that direct the initiation of cotyledon primordia (Reinhardt et al. 2003, Hay et al. 2004, Jenik and Barton 2005). Arabidopsis possesses 23 ARF proteins that can bind to the auxin-responsive cis-acting elements of their target genes. Aux/IAA proteins are encoded by 29 genes in Arabidopsis and negatively modulate auxin-responsive gene expression through heterodimerization with ARF proteins (Tiwari et al. 2001, Tiwari et al. 2003, Tiwari et al. 2004). Auxin-dependent degradation of Aux/IAA proteins releases ARF from their interactions with these proteins and allows the activation or repression of target genes (reviewed in Guilfoyle and Hagen 2007, Chapman and Estelle 2009). Two members of the ARF gene family, MP and NONPHOTOTROPIC HYPOCOTYL 4 (NPH4)/ARF7, and the Aux/IAA genes, BODENLOS (BDL)/IAA12 and IAA13, have been shown to have a role in embryo axis formation including cotyledon initiation (Berleth and Jürgens 1993, Przemeck et al. 1996, Hamman et al. 1999, Hamman et al. 2002, Hardtke et al. 2004, Weijers et al. 2005, Weijers et al. 2006). However, it is still possible that other members of these protein families contribute to cotyledon initiation, and little is known of the molecular mechanisms responsible for auxin-responsive transcription via ARFs.

Although auxin maxima are altered in pid mutants, much of the embryo patterning occurs normally, indicating the existence of other controls (Treml et al. 2005, Furutani et al. 2007). To identify these regulators, especially those involved in cotyledon organogenesis mediated by auxin, we screened the pid enhancer mutant macchi-bou (mab) (Furutani et al. 2007). pid mutants show mild phenotypes including effects on cotyledon number and positioning (Bennett et al. 1995, Christensen et al. 2000, Benjamins et al. 2001), whereas mab mutants completely lack cotyledons on a pid background (Furutani et al. 2007). Recently we succeeded in identifying and characterizing the MAB4 gene, which encodes a novel NPH3-like protein. MAB4 is identical to ENHANCER of PINOID and its function is required for control of PIN1 polarity (Treml et al. 2005, Furutani et al. 2007).

In this study, we identified and characterized a novel Arabidopsis gene, MAB2, which encodes a homolog of Mediator complex subunit 13 (MED13) and is identical to GRAND CENTRAL (GCT) (Gillmor et al. 2010). The mab2 mutant shows disturbance of the cell division pattern at an early stage of embryogenesis and causes aberrant cotyledon development. Analysis of auxin markers and investigation of the genetic interaction with auxin-insensitive mutants revealed that MAB2 is required for auxin response at least in the apical region of embryos. These results indicate that MAB2 is involved in the control of cell division patterns and of cotyledon primordia formation possibly through transcriptional regulation of specific genes such as auxin-responsive genes.

Results

mab2-1 is a new pid enhancer mutant

To investigate the mechanism of cotyledon organogenesis, we screened ethylmethane sulfonate (EMS)-mutagenized pid-2 lines and isolated a mutant that displayed severe defects in cotyledon development (Fig. 1C); we named this mutation mab2. While pid-2 mutant seedlings showed only a mild phenotype in cotyledon positioning, number and separation compared with the wild type (Fig. 1A, B) (Bennett et al. 1995), mab2-1 pid-2 double mutants completely lacked cotyledons (Fig. 1C).

Fig. 1

Morphological defects in mab2-1 mutants. (A–G) Seven-day-old seedlings of the wild type (A), pid-2 (B), mab2-1 pid-2 (C) and mab2-1 (D–G). Arrowheads in C and G indicate the absence of cotyledons. (H) mab2-1 adult plant at 70 d post-germination. (I–K) Open siliques of the wild type (I) and mab2-1/+ (J). (K) An enlarged image of the region enclosed within the white square in J. White arrows indicate pale green and shriveled seeds. The red arrow in H indicates the leaf-like structure at the basis of the pedicel. Scale bars = 250 μm.

Fig. 1

Morphological defects in mab2-1 mutants. (A–G) Seven-day-old seedlings of the wild type (A), pid-2 (B), mab2-1 pid-2 (C) and mab2-1 (D–G). Arrowheads in C and G indicate the absence of cotyledons. (H) mab2-1 adult plant at 70 d post-germination. (I–K) Open siliques of the wild type (I) and mab2-1/+ (J). (K) An enlarged image of the region enclosed within the white square in J. White arrows indicate pale green and shriveled seeds. The red arrow in H indicates the leaf-like structure at the basis of the pedicel. Scale bars = 250 μm.

To investigate the MAB2 function, mab2-1 single mutants were isolated and an analysis of mab2-1 phenotypes was carried out. The mab2-1 mutation caused aberrant cotyledon development and embryo lethality. As homozygous mab2-1 plants were sterile, we analyzed seedling phenotypes in homozygous mutants segregating from self-fertilized heterozygous mab2-1 plants. The mab2-1 homozygotes showed a variety of phenotypes: almost half had two separate symmetrical cotyledons as in the wild type (Fig. 1E, Table 1); others showed defective cotyledon development, including mono- and tricotyledonous phenotypes (Fig. 1D, F, Table 1); and some developed two bulges instead of normal cotyledons (Fig. 1G, Table 1). The self-fertilized heterozygous mab2-1 plants produced only 4.7% mutant seedlings (Table 1). This rate of homozygous mutant production is considerably smaller than the expected 25%, suggesting that approximately 80% of the homozygous mab2-1 mutants were lost through embryonic lethality. We found that 7% of the seeds in siliques of mab2-1 heterozygous plants were aborted with a small and shriveled appearance, and 15% were rudimentary (n = 329); in contrast, 3% in the wild type were either shriveled or rudimentary (n = 416) (Fig. 1I–K).

Table 1

Frequency of cotyledon phenotypes segregating from mab2-1/+

Genotype Frequency of cotyledon number (%)
 
Total numbers of seedlings (segregation rate) 
 Zero One Two Three  
mab2-1/mab2-1 12.9 12.9 61.3 12.9 31 (4.7%) 
mab2-1/+ 3.3 96.8 400 (60.4%) 
MAB2/MAB2 0.4 0.9 98.7 233 (34.9%) 
Genotype Frequency of cotyledon number (%)
 
Total numbers of seedlings (segregation rate) 
 Zero One Two Three  
mab2-1/mab2-1 12.9 12.9 61.3 12.9 31 (4.7%) 
mab2-1/+ 3.3 96.8 400 (60.4%) 
MAB2/MAB2 0.4 0.9 98.7 233 (34.9%) 

We genotyped 664 seedlings segregating from mab2-1 heterozygous seeds and classified the cotyledon phenotypes according to the cotyledon number.

The mab2-1 mutants displayed various defects in post-embryonic development. Although some seedlings died before bolting, the remainder continued to develop after germination. These plants had a greatly delayed flowering time. As shown in Fig. 1H, mab2-1 adult plants had a slightly bushy architecture and also produced sterile flowers that had an ectopic leaf-like structure at the base of each pedicel. We could not detect any obvious abnormalities in the identity and the number of floral organs of homozygous mab2-1 plants (data not shown).

Embryogenesis in the mab2 mutant

To determine the developmental origin of the cotyledon defect and embryo lethality, we analyzed whole-mount preparations of developing seeds after self-fertilization in heterozygous mab2-1 plants (Fig. 2). Embryos with phenotypic deviation from the wild type were first identified at the 4- to 8-cell stage: 11.3% (6/53) of the embryos had upper suspensor cells that divided vertically instead of horizontally as in the wild type (Fig. 2A–D). At the globular stage, the embryo proper also divided abnormally but retained the round shape at the apical region (Fig. 2E, F). At the subsequent heart stage, some mab2-1 embryos continued to undergo abnormal cell division and formed a disordered cell cluster (Fig. 2G, H). Another defect also became apparent at this stage. Approximately, 13% (23/173) of the embryos either lacked or had an aberrant number of cotyledon primordia (Fig. 2G, I–L). Although some mutant embryos with the correct number of cotyledon primordia were observed, these displayed asymmetric development or developmental retardation of the cotyledon primordia (Fig. 2M). Thus, the mab2-1 mutation caused defects in cell division and in development of cotyledon primordia at an early stage of embryogenesis. These findings suggest that the mab2-1 seedling phenotypes originate during embryogenesis.

Fig. 2

Defects in embryogenesis in mab2-1 mutants. Wild type or heterozygous mutant embryos (A, C, E, G, K) and homozygous mab2-1 mutants (B, D, F, H–J, L, M). (A, B) Four-cell stage, (C, D) 8-cell stage, (E, F) globular stage, (G–J) heart stage, (K–M) torpedo stage. Arrows indicate cells that have undergone abnormal division. Arrowheads mark the positions of cotyledon primordia. Scale bars = 20 μm.

Fig. 2

Defects in embryogenesis in mab2-1 mutants. Wild type or heterozygous mutant embryos (A, C, E, G, K) and homozygous mab2-1 mutants (B, D, F, H–J, L, M). (A, B) Four-cell stage, (C, D) 8-cell stage, (E, F) globular stage, (G–J) heart stage, (K–M) torpedo stage. Arrows indicate cells that have undergone abnormal division. Arrowheads mark the positions of cotyledon primordia. Scale bars = 20 μm.

Expression of PIN1:GFP and DR5rev::GFP in mab2-1 embryos

The defective cell division and cotyledon development during embryogenesis in mab2-1 mutants is reminiscent of the embryonic disturbances observed in auxin-related mutants. Additionally, the seedling phenotypes of the mab2-1 pid double mutant were almost identical to those of pin1 pid and mab4 pid mutants (Furutani et al. 2004, Furutani et al. 2007), suggesting that MAB2 is involved in auxin-regulated organogenesis. To investigate the relationship between MAB2 and auxin action, we analyzed PIN1:GFP (green fluorescent protein) expression in mab2-1 embryos to determine the effect of the mutation on auxin transport. Although mab2-1 mutants had altered cotyledon numbers, expression of PIN1:GFP in the cotyledon primordia was similar to that in the wild type, and its localization at the plasma membrane was also the same as in the wild type (Fig. 3A, B, E, F). Next, we used DR5rev::GFP, which indirectly monitors auxin response (Friml et al. 2003), to examine the distribution of auxin in mab2-1 embryos. In wild-type embryos at the heart stage, DR5rev::GFP maxima were observed at the tips of the developing cotyledons and at the base of the embryo (Fig. 3C). In contrast, DR5rev::GFP signals were often missing or weak at the developing cotyledons of mab2-1 embryos, whereas signals equivalent to those of wild-type embryos were present at the basal region of mab2-1 embryos (Fig. 3D). These expression patterns were maintained in the subsequent torpedo stage embryos (Fig. 3G, H).

Fig. 3

Auxin response is altered in mab2-1 embryos. (A, B, E, F) PIN1:GFP localization in wild-type (A, E) and mab2-1 embryos (B, F). (C, D, G, H) DR5rev::GFP expression in wild type (C, G) and mab2-1 embryos (D, H). (A–D) Heart stage embryos. (E–H) Torpedo stage embryos. White arrowheads indicate the positions of auxin response. Scale bars = 10 μm.

Fig. 3

Auxin response is altered in mab2-1 embryos. (A, B, E, F) PIN1:GFP localization in wild-type (A, E) and mab2-1 embryos (B, F). (C, D, G, H) DR5rev::GFP expression in wild type (C, G) and mab2-1 embryos (D, H). (A–D) Heart stage embryos. (E–H) Torpedo stage embryos. White arrowheads indicate the positions of auxin response. Scale bars = 10 μm.

Genetic interaction between mab2-1 and auxin-response mutants

To test for genetic interaction between mab2-1 and auxin-response mutants, we created a double mutant using a dominant gain-of-function allele for the Aux/IAA gene, BDL (Hamann et al. 1999). Seven-day-old wild-type seedlings are comprised of two separated cotyledons, primary leaves, a hypocotyl and a root, in all of which continuous vascular bundles are arranged (Fig. 4A–C). In contrast, 7-day-old mab2-1 seedlings possessed aberrant cotyledons with an incomplete venation patterning (Fig. 4D–F); however, their hypocotyl and primary root formation, including the vasculature, was fairly normal (Figs. 1D–G, 4D, E). As described by Hamann et al. (1999), the bdl mutation yields seedlings without roots and produces two phenotypic classes in a single mutant allele. Mutant seedlings with the weaker phenotype lacked most of the primary root and the root meristem (Fig. 4G, J) but they did harbor continuous vascular strands (Fig. 4H–J). The stronger bdl phenotype lacked a hypocotyl, root and primary root meristem (Fig. 4K, N), and displayed reduced vasculature (Fig. 4L–N). The mab2-1 mutation caused a remarkable enhancement of bdl seedling phenotypes. Regardless of the bdl phenotype strength, mab2-1 bdl double mutant seedlings were completely devoid of cotyledons and continuous vascular strands, as well as lacking a root (Fig. 4O–S). The SAM in the mab2-1 bdl double mutant was present above a cluster of vessel elements and often formed multiple bulges and primary leaf-like structures (Fig. 4O–Q). We occasionally found seedlings with two SAMs at the apical region (Supplementary Fig. S1). The basal part of mab2-1 bdl seedlings failed to form any root architecture, similar to bdl seedlings, and thereby resembled a basal peg structure (Fig. 4S). We performed another test for genetic interaction using mab2-1 and the auxin-response mutant mp (Berleth and Jürgens 1993). mp mutants are also rootless like bdl although their various alleles can show phenotypic differences in vascular defects (Berleth and Jürgens 1993, Hardtke et al. 2004). The mpT370 mutant used in this study is classified as an intermediate mp allele and shows an extremely reduced vascular system (Supplementary Fig. S2A–C). Similar to the mab2-1 bdl double mutant seedlings described above, mab2-1 mpT370 seedlings failed to form cotyledons and to show differentiation of aligned vascular cells (Supplementary Fig. S2D–F). The basal part of the mab2-1 mpT370 mutant seedling was comprised of a basal peg structure (Supplementary Fig. S2G). Thus, the two double mutant phenotypes investigated here suggest that a synergistic interaction occurred between mab2-1 and auxin-response mutants.

Fig. 4

Synergistic interaction of the mab2-1 bdl mutations. Seven-day-old seedlings of the wild type (A–C), mab2-1 (D–F), bdl with the weaker phenotype (G–J), bdl with the stronger phenotype (K–N) and mab2-1 bdl (O–S). (C, F, I, M, Q) Vascular strands at the basal end of cotyledons in wild type (C), mab2-1 (F), bdl (I, M) and mab2-1 bdl (Q). (R) An enlarged image of the region enclosed within the black square in Q. (J, N) The basal end of seedlings with or without vascular strands and root hair in bdl. (S) The basal peg in mab2-1 bdl. (A, D, G, K, O) Live seedlings. (B, E, H, L, P) Dark-field images of cleared seedlings. Scale bars = 0.5 mm. (C, F, I, J, M, N, Q–S) Nomarski images of cleared seedlings. Scale bars = 50 μm.

Fig. 4

Synergistic interaction of the mab2-1 bdl mutations. Seven-day-old seedlings of the wild type (A–C), mab2-1 (D–F), bdl with the weaker phenotype (G–J), bdl with the stronger phenotype (K–N) and mab2-1 bdl (O–S). (C, F, I, M, Q) Vascular strands at the basal end of cotyledons in wild type (C), mab2-1 (F), bdl (I, M) and mab2-1 bdl (Q). (R) An enlarged image of the region enclosed within the black square in Q. (J, N) The basal end of seedlings with or without vascular strands and root hair in bdl. (S) The basal peg in mab2-1 bdl. (A, D, G, K, O) Live seedlings. (B, E, H, L, P) Dark-field images of cleared seedlings. Scale bars = 0.5 mm. (C, F, I, J, M, N, Q–S) Nomarski images of cleared seedlings. Scale bars = 50 μm.

Identification of the MAB2 gene

We isolated the MAB2 gene using map-based cloning. The MAB2 locus is located between molecular markers F7A10 and T5A14 on chromosome 1 (Fig. 5A). We compared the sequences of several putative genes predicted to be located within this region in wild-type and mab2-1 mutant genomes. In the mab2-1 mutant, a G to A nucleotide transition at amino acid position 1,833 was found in the open reading frames of At1g55325; this change causes the substitution of a tryptophan with a stop codon to delete 45 amino acids at the C-terminal region (Fig. 5A, Supplementary Fig. S4C). Recently, this gene was identified as GCT and is involved in the regulation of peripheral–abaxial polarity during early embryogenesis (Gillmor et al. 2010). A reverse transcription–PCR (RT–PCR) analysis showed that the expression level of At1g55325 was not changed in the mab2-1 mutant seedlings compared with that of the wild type (Supplementary Fig. S4B). To confirm that the MAB2 gene is identical to At1g55325, we introduced At1g55325 cDNA, driven by a 2.0 kb upstream region, into plants heterozygous for mab2-1. From these transformed plants, we then obtained transgenic plants homozygous for the mab2-1 mutation; these plants produced normal flowers without a leaf-like structure at the base of the pedicel and also showed partially restored embryo development (Supplementary Fig. S3A).

Fig. 5

The MAB2 gene encodes MED13. (A) Map-based cloning of the MAB2 gene. The MAB2 locus was mapped to the region between the F7A10 and T5A14 markers on chromosome 1. The mab2-1 mutation is a single base substitution in At1g55325. T-DNA in mab2-2, 2-3, 2-4 mutants was inserted into the third exon, the eighth intron and the tenth exon, respectively. (B) Comparison of the deduced protein sequence of the TRAP240 domain of MAB2, OsMED13 (GenBank accession No. EAZ34492), PtMED13 (GenBank accession No. EEE79832), HsMED13/TRAP240 (GenBank accession No. EAW51438), DrMed13 (GenBank accession No. AAI29033), DmMED13/Skuld (GenBank accession No. AAG48327), CeTRAP240/LET-19 (GenBank accession No. BAE16563) and SSN9 (GenBank accession No. Q9HE02). Alignment was carried out with the DDBJ program (CLUSTAL W, version 1.83).

Fig. 5

The MAB2 gene encodes MED13. (A) Map-based cloning of the MAB2 gene. The MAB2 locus was mapped to the region between the F7A10 and T5A14 markers on chromosome 1. The mab2-1 mutation is a single base substitution in At1g55325. T-DNA in mab2-2, 2-3, 2-4 mutants was inserted into the third exon, the eighth intron and the tenth exon, respectively. (B) Comparison of the deduced protein sequence of the TRAP240 domain of MAB2, OsMED13 (GenBank accession No. EAZ34492), PtMED13 (GenBank accession No. EEE79832), HsMED13/TRAP240 (GenBank accession No. EAW51438), DrMed13 (GenBank accession No. AAI29033), DmMED13/Skuld (GenBank accession No. AAG48327), CeTRAP240/LET-19 (GenBank accession No. BAE16563) and SSN9 (GenBank accession No. Q9HE02). Alignment was carried out with the DDBJ program (CLUSTAL W, version 1.83).

We obtained other mab2 alleles, mab2-2/gct-5, mab2-3 and mab2-4, that carry a T-DNA insertion in At1g55325 (Fig. 5A) (Alonso et al. 2003). These mutants displayed the same phenotypes as mab2-1, including aberrant cotyledon development, ectopic leaf-like formation at the base of the pedicel, and sterility (Supplementary Fig. S4A). When mab2-2/gct-5 that has a T-DNA insertion at the third exon of At1g55325 was crossed with a pid-3 mutant, cotyledons were completely absent (Supplementary Fig. S4A). Next, we crossed plants heterozygous for mab2-1 with mab2-2/gct-5 heterozygotes and analyzed the F1 plants carrying both mutations. The F1 plants had a flower subtended by a leaf-like structure and did not show complementation of the mutant phenotype (Supplementary Fig. S3B). Taken together, these results led us to conclude that MAB2 corresponds to At1g55325.

MAB2 is a homolog of MED13

MAB2 encodes a protein of 1,877 amino acids that has many properties in common with MED13. The MAB2 protein contains a thyroid hormone receptor-associated protein (TRAP) 240 domain, known to be a conserved domain among MED13 proteins in organisms such as yeast, Drosophila and humans (Fig. 5B). In addition, all 27 signature sequence motifs (SSMs) of MED13, defined by Bourbon (2008), are conserved in MAB2 (Supplementary Fig. S4C). MED13 is a component of the cyclin-dependent kinase 8 (CDK8) subcomplex. The CDK8 subcomplex consists of CDK8, its C-type cyclin (CycC) partner, MED12 and MED13; the subcomplex functions as a separable accessory module of Mediator, a conserved multisubunit complex bridging transcriptional regulators to the RNA polymerase II initiation machinery (Malik and Roeder 2005). Homologs of the Mediator complex, including the CDK8 subcomplex, have been identified in Arabidopsis (Bäckström et al. 2007, Bourbon 2008) and in a wide range of other plant species, including angiosperms (Oryza sativa, Populus trichocarpa), bryophytes (Physcomitrela patens) and chlorophyta (Chlamydomonas reinhardtii) (Bourbon 2008).

Interaction between Arabidopsis CDK8 subunits

CENTER CITY (CCT)/CRYPTIC PRECOCIOUS (CRP) and HUA ENHANCER3 (HEN3) are the Arabidopsis homologs of MED12 and CDK8, respectively (Wang and Chen 2004, Gillmor et al. 2010). Additionally, the Arabidopsis genome appears to contain two CycC homologs, CYCC1;1 and CYCC1;2 (Wang et al. 2004), suggesting the existence of a CDK8 subcomplex in Arabidopsis. To examine the possibility of direct interaction between Arabidopsis CDK8 submodules, we performed a yeast two-hybrid assay. The yeast MED13 homolog Srb9 has been reported to show transcriptional activation by itself when expressing the Srb9 fusion protein with the DNA-binding domain (BD) (Guglielmi et al. 2004) and, in Drosophila, interaction between the CDK8 submodules was detected only when MED13 was fused with the DNA activation domain (AD) (Loncle et al. 2007). Therefore, as shown in Fig. 6, we prepared yeast strains expressing the MAB2 protein fused with the AD and other submodules fused with the BD, and other strains expressing the HEN3 protein fused with the AD and other submodules fused with the BD. Using a HIS3 reporter gene activity assay, we found that MAB2 interacted only with CYCC1;2 in the yeast cells. Interaction between CYCC1;2 and HEN3 was also detected. These interactions were confirmed by an assay using the lacZ reporter gene, and were detected at similar levels to the interaction between positive controls.

Fig. 6

MAB2 interacts with CYCC1;2. MAB2 and HEN3 were expressed as fusion proteins with a transcriptional activator domain (AD), and HEN3, CCT/CRP, CYCC1;1 and CYCC1;2 were expressed as fusion proteins with a DNA-binding domain (BD). Interaction between two proteins was tested using the HIS3 reporter gene and the lacZ reporter gene, respectively. The presence of β-galactosidase activity in LacZ+ colonies was detected 1 h after incubation at 30°C in this experiment. pBD-wt and pAD-wt were used as positive controls (cont).

Fig. 6

MAB2 interacts with CYCC1;2. MAB2 and HEN3 were expressed as fusion proteins with a transcriptional activator domain (AD), and HEN3, CCT/CRP, CYCC1;1 and CYCC1;2 were expressed as fusion proteins with a DNA-binding domain (BD). Interaction between two proteins was tested using the HIS3 reporter gene and the lacZ reporter gene, respectively. The presence of β-galactosidase activity in LacZ+ colonies was detected 1 h after incubation at 30°C in this experiment. pBD-wt and pAD-wt were used as positive controls (cont).

Expression patterns of MAB2

The tissue specificity of MAB2 expression was examined by quantitative RT–PCR analysis. As shown in Fig. 7A, MAB2 mRNA was detected in all tissues examined. This result is consistent with other published microarray data (Zimmermann et al. 2004, Winter et al. 2007). In order to investigate the spatial distribution of the MAB2 gene in wild-type embryos, we performed an in situ hybridization analysis. In wild-type embryos, MAB2 expression was detected ubiquitously at all developmental stages (Fig. 7B–F). MAB2 mRNA was also expressed in the suspensor and the endosperm (Fig. 7B, C). No signals were detected in the embryo using a control sense probe (Fig. 7G).

Fig. 7

Expression analysis of MAB2. (A) Expression levels of MAB2 in various tissues. The expression levels were normalized against β-tubulin. Data shown are means of three independent samples, with error bars representing the SD. (B–G) MAB2 mRNA accumulation in wild-type embryos at the 8-cell stage (B), 16-cell stage (C, G), globular stage (D), heart stage (E) and late heart stage (F). (G) No hybridization was detected with the sense control probe. Arrowheads indicate endosperm expressing MAB2 mRNA. Scale bars = 10 μm.

Fig. 7

Expression analysis of MAB2. (A) Expression levels of MAB2 in various tissues. The expression levels were normalized against β-tubulin. Data shown are means of three independent samples, with error bars representing the SD. (B–G) MAB2 mRNA accumulation in wild-type embryos at the 8-cell stage (B), 16-cell stage (C, G), globular stage (D), heart stage (E) and late heart stage (F). (G) No hybridization was detected with the sense control probe. Arrowheads indicate endosperm expressing MAB2 mRNA. Scale bars = 10 μm.

Discussion

In this study, we used a pid enhancer screen to identify a novel gene, MAB2, which is involved in cotyledon organogenesis. MAB2 encodes a transcriptional regulatory module of Mediator, MED13, and mutation of the gene causes severe defects in early embryo patterning and cotyledon organogenesis, perhaps as a consequence of the reduction in auxin response. Our data suggest that MAB2 functions as a key modulator for transcription to regulate correct embryo patterning.

MAB2 is required for correct embryo patterning

In Arabidopsis, cotyledons develop during embryogenesis. Although the MAB2 gene is expressed throughout embryogenesis (Fig. 7B–F), mutation of the gene principally affected two different stages: (i) cell division patterning from the onset of the 4- to 8-cell stage, presumably resulting in embryo lethality as shown in Fig. 2H; and (ii) development of cotyledon primordia from the globular to heart stage (Fig. 2I, J). These defects open up the possibility of correlation between MAB2 function and auxin action. Auxin is a prerequisite for the establishment of the correct embryo patterning (Benková et al. 2003, Friml et al. 2003, Blilou et al. 2005, Vieten et al. 2005). Directional auxin flow is mediated by polar PIN1 localization to create auxin maxima at the predicted sites of organ primordia, resulting in the initiation of organogenesis through activation of specific ARF genes (Reinhardt et al. 2003, Jenik et al. 2007, Bowman and Floyd 2008). It is noteworthy that abnormal cell division at the boundary region between embryo and suspensor in the mab2 mutant was also observed in embryos of auxin-response mutants such as mp (Fig. 2; Bennett et al. 1995). Additionally, in mab2 embryos, expression of the auxin-responsive marker DR5rev::GFP was impaired in the developing cotyledons similar to that seen in auxin-response mutants (Fig. 3). Since directional auxin flow might be established by correct PIN1 localization (Fig. 3), it appears that a reduced auxin response at the apical portion of mab2 embryos contributes to defects in cotyledon development. These findings suggest that impaired auxin response in mab2 embryos causes the morphological defects in mab2-1 mutants.

How MAB2 acts at the molecular level is still not understood. The synergistic effects in the mab2-1 bdl double mutant seedlings indicate that the MAB2 and BDL genes function in the same processes such as embryo patterning and vascular development (Fig. 4). BDL interacts with MP to inhibit the activation potential of MP in embryogenesis (Hamann et al. 2002, Tiwari et al. 2004, Weijers et al. 2005). MP activity is required for embryo patterning, especially hypophysis formation and cotyledon development during embryogenesis (Hardtke and Berleth 1998, Hardtke et al. 2004, Weijers et al. 2006). Furthermore, MP is expressed in the developing vasculature and functions as a limiting factor in expression of the leucine zipper transcription factors AtHB8 and AtHB20 that are associated with procambial development (Hardtke and Berleth 1998, Mattson et al. 2003). Mutation of the MP gene results in seedling phenotypes similar to those of the gain-of-function bdl mutant (Hamann et al. 2002), and the mab2-1 mutation also enhances the phenotypes of mpT370 seedlings (Supplementary Fig. S2). The mp bdl double mutant is devoid of cotyledons, similar to mab2-1 bdl and mab2-1 mpT370 double mutants (Hardtke and Berleth 1998). Along the same lines, the defects in mab2-1 bdl and mab2-1 mpT370 double mutants are also reminiscent of those in the strong mp nph4/arf7 double mutant seedlings, which completely lack cotyledons and continuous vascular strands, resulting in ‘club-shaped’ seedlings (Hardtke et al. 2004). Both MP and NPH4 are capable of self-interaction and interacting with each other, and their expression profiles overlap in embryos, suggesting that MP and NPH4 may have redundant functions in embryogenesis (Hardtke et al. 2004). In this context, the mutant phenotypes described above support the notion that MAB2 might share its role in embryo patterning with MP, NPH4 and BDL, which regulate auxin distribution and signaling. However, the phenotypes of mab2 mutants are not completely consistent with those of mp mutants. Some mab2 mutants can form an embryonic root meristem while mp mutants fail to do so (Figs. 1, 2, Supplementary Fig. S2). The auxin response at the basal region of the embryos differs between these two mutants. Expression of the DR5rev::GFP reporter in the hypophysis was absent in mp embryos although it was fully active at the corresponding region in mab2-1 embryos (Fig. 3D, H). These facts indicate that MAB2 does not contribute to embryonic root formation.

Moreover, the broad expression pattern of MAB2 mRNA in embryos implies another regulation of embryo patterning by MAB2 besides auxin action. gct mutants, another mab2 allele, were identified in a genetic screen using the KANADI2 (KAN2) enhancer trap line (Gillmor et al. 2010). KAN is required for specification of abaxial identity in leaves and carpels and for the peripheral identity in the developing embryo (Kerstetter et al. 2001). In gct mutant embryos, a developmental delay in embryo patterning and disordered cell patterning were observed as in mab2 embryos. Gillmor et al. (2010) showed that these defects could be attributed to a delay in KAN expression at early embryogenesis and also in expression of genes such as SHOOT MERISTEMLESS (STM) that are required for the formation and maintenance of the SAM at subsequent stages of embryogenesis. That is, GCT/MAB2 might regulate the spatial and temporal expression of specific genes during embryogenesis. Further studies, in particular for identification of direct targets of MAB2, will help find the precise roles of MAB2 during embryogenesis.

MAB2 encodes AtMED13, a regulatory module of the Mediator complex

We have shown that MAB2 encodes an Arabidopsis ortholog of MED13 and that its deduced protein contains a TRAP240 domain and 27 SSMs. The Arabidopsis genome contains a single copy of the MAB2 gene. In yeast and animal cells, MED13 interacts both physically and functionally with MED12, CDK8 and CycC to form a specific regulatory module of the Mediator, the CDK8 subcomplex (Borggrefe et al. 2002, Taatjes et al. 2004, Andrau et al. 2006). The CDK8 subcomplex is known to associate with Mediator to block interactions with RNA polymerase II, thereby providing a negative control of transcription (Malik and Roeder 2005). Conversely, recent studies using genetic and genome-wide analyses clearly showed that the CDK8 subcomplex could also function as an active regulator of transcription (Larschan and Winston 2005, Andrau et al. 2006, Zhu et al. 2006, Donner et al. 2007, Carrera et al. 2008), although the molecular mechanism of this process remains unclear. Homologs of Mediator and the CDK8 subcomplex show evolutionary conservation in eukaryotes including Arabidopsis (Bäckström et al. 2007, Bourbon 2008). These findings raise the possibility that MAB2 interacts with other submodules of the CDK8 subcomplex to regulate the transcription of various genes. We found that CYCC1;2 physically interacted with MAB2 and with HEN3 in a yeast two-hybrid assay (Fig. 6). These results suggest that MAB2 can interact directly with CYCC1;2 and indirectly with HEN3. However, we failed to detect any interaction between CYCC1;1 and other submodules. At present, the differences between CYCC1;1 and CYCC1;2 are unclear. Although CCT/CRP did not interact with any CDK8 submodules in yeast cells, a functional relationship between CCT/CRP and MAB2 has been reported. Both CCT/CRP and MAB2/GCT are required for KAN expression, and the phenotypes of the cct/crp and mab2/gct mutants including expression pattern of auxin markers in their embryos (data not shown) are very similar (Gillmor et al. 2010). These results strongly suggest that MAB2/GCT and CCT/CRP possess closely related functions and work in the same pathway.

The Mediator complex in plant development

The mab2-1 mutation also affected post-embryonic development, such as delaying flowering time, causing ectopic formation of a leaf-like structure at the basal position of the flower, and sterility. Expression analysis by quantitative RT–PCR showed that MAB2 was highly expressed in the aerial organs. Interestingly, mutation of MAB2 affected specific developmental processes. This finding is consistent with previous studies that reported that MED13 and MED12 are required for specific developmental processes in Drosophila, zebrafish and Caenorhabditis elegans (Treisman 2001, Janody et al. 2003, Hong et al. 2005, Yoda et al. 2005, Rau et al. 2006, Loncle et al. 2007, Clayton et al. 2008). However, it is difficult to explain that all of the phenotypic effects in mab2 mutants are due to auxin action, suggesting that MAB2 might also be involved in the auxin-independent developmental program. Previously, four Mediator subunits have been described in Arabidopsis. STRUWWELPETER is a homolog of MED14 and involved in the regulation of cell proliferation at the meristem (Autran et al. 2002, Bäckström et al. 2007). PHYTOCHROME AND FLOWERING TIME 1 (PFT1), which is well known as a positive regulator of FLOWERING LOCUS T via phytochrome B (Cerdán and Chory 2003), was identified as the MED25 subunit by Bäckström et al. (2007). More recently, PFT1 and another Mediator subunit, MED8, have been shown to be required for both plant jasmonate-dependent defense and flowering time regulation (Kidd et al. 2009). Similarly, it was shown that the MED21 subunit is also required for resistance to necrotrophic pathogens in Arabidopsis (Dhawan et al. 2009). Mutation of MAB2 affected the flowering time like pft1 and med8. This observation opens up the possibility that interaction of the core Mediator and CDK8 subcomplex controls the expression of flowering time regulation genes. Overall, these findings indicate that plant Mediator subunits, as well as MAB2, play a role as important transcriptional regulators depending on the target genes and the developmental context, rather than as components of the general transcriptional machinery. Future research aimed at identification and characterization of functional units in Mediator will help to advance our understanding of transcriptional regulation in plants.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana ecotypes Landsberg erecta (Ler) and Columbia (Col-0) were used as wild types. The mutant alleles used in this study were pid-2 (Ler) (Christensen et al. 2000), pid-3 (Col) (Bennett et al. 1995), bdl (Ler) (Hamann et al. 1999) and mpT370 (Ler) (Berleth and Jürgens 1993). mab2-1 was isolated in our laboratory, and originated from EMS-mutagenized lines of pid-2. mab2-2/gct-5 (SAIL_1169_H11), mab2-3 (SAIL_413_A07) and mab2-4 (SAIL_510_A12) were obtained from the Arabidopsis Biological Resource Center and were backcrossed three times to Col-0 before any analysis and construction of the double mutants. Seeds were surface sterilized, plated on MS (Murashige and Skoog) medium plates, and germinated as previously described (Fukaki et al. 1996). Plants were transferred to soil and were grown at 23°C under constant light as described previously (Fukaki et al. 1996).

Microscopy

Cleared whole-mount samples were prepared as described in Berleth and Jürgens (1993). Fluorescence images were acquired by confocal laser scanning microscopy (FV1000; Olympus). For confocal microscopy, dissected embryos were mounted in 7% glucose.

Mapping and cloning of the MAB2 gene

The mab2-1 mutant was crossed with pid-3/+ or Col. Among the F2 populations, seedlings without cotyledons or plants with flowers subtended by a leaf-like structure were used for map-based cloning. The MAB2 locus was eventually mapped between two simple sequence length polymorphism markers, F7A10 and T5A14, on chromosome 1. The genomic sequences of the MAB2 locus were amplified by PCR. The resulting PCR products were directly sequenced using a BigDye terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3100 sequencer (Applied Biosystems).

For the complementation test, the 2.0 kb upstream region of At1g55325 and MAB2 cDNA, which included the 180 bp of the 5′ untranslated region (UTR), the 5.6 kb of the coding region and the 356 bp of the 3′ UTR were cloned into pGWB1 (Nakagawa et al. 2007). This construct was transformed into Agrobacterium tumefaciens strain GV3101::pMP90, and was then introduced into plants heterozygous for the mab2-1 mutation using the floral dipping method (Clough and Bent 1998).

Allelism test

To examine allelism in MAB2 and At1g55325/GCT, mab2-1/+ plants were crossed to heterozygous plants with a T-DNA insertion into At1g55325 (gct-5/SAIL_1169_H11). The resulting F1 progeny, which carry each gene heterozygously, were screened to determine whether the presence of both mutations resulted in complementation of the mab2-1 mutant phenotype.

Quantitative RT–PCR

Quantitative RT–PCR was performed according to Igari et al. (2008). PCR was performed using the following primer sets: MAB2, MAB2-R1 (5′-TATGCCCGGATATTGATCCT-3′) and MAB2-L1 (5′-GCACGTTTCATAAACAGTTCCA-3′); and β-tubulin, β-tubulin-4F, (5′-GAGGGAGCCATTGACAACATCTT-3′) and β-tubulin-4R, (5′-GCGAACAGTTCACAGCTATGTTCA-3′).

In situ hybridization

In situ hybridization was performed as described by Takada et al. (2001). Hybridization was carried out at 45°C. Western Blue (Promega) was used as the substrate for signal detection. To generate a MAB2 probe, a 798 bp fragment corresponding to a region of MAB2 cDNA was amplified from 7-day-old seedlings using the following primers: cMAB2 12f, 5′-GGTGACTTCTTTGAGAACGATGC-3′ and cMAB2 12r, 5′-AGCTTGGAACATAACACTTC-3′.

Yeast two-hybrid assay

MAB2 and HEN3 cDNAs were subcloned into pAD-GAL4-GWRFC (Yamaguchi et al. 2008). CCT/CRP, HEN3, CYCC1;1 and CYCC1;2 cDNAs were subcloned into pBD-GAL4-GWRFC (Yamaguchi et al. 2008). The resultant plasmids were introduced into the AH109 strain using a Fast™-Yeast Transformation kit (G-Biosciences). The empty vectors were used as negative controls. pBD-wt and pAD-wt (Stratagene) were used as positive controls. Interaction between the bait and target proteins was detected by the expression of the HIS3 reporter gene. To distinguish between leaky expression of the HIS3 gene and specifically interacting proteins, detection of a second reporter gene (lacZ) was determined using the filter lift assay.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology [the Global COE Program A9 to M.T. and M.F, 14036222 to M.T and 20770034 to M.F].

Acknowledgments

We thank Gerd Jüergens for providing bdl seeds, Taku Demura for providing pAD-GWRFC and pBD-GWRFC vectors, and Ms. Asami Mori for technical assistance. We also thank the Arabidopsis Biological Resource Center for providing T-DNA insertion mutant lines.

Abbreviations

    Abbreviations
  • AD

    DNA activation domain

  • ARF

    AUXIN RESPONSE FACTOR

  • Aux/IAA

    Auxin/IAA

  • BD

    DNA-binding domain

  • BDL

    BODENLOS

  • CCT

    CENTER CITY

  • CDK

    cyclin-dependent kinase

  • CycC

    cyclin C

  • EMS

    ethylmethane sulfonate

  • GCT

    GRAND CENTRAL

  • GFP

    green fluorescent protein

  • HUA

    HUA ENHANCER

  • MAB

    MACCHI-BOU

  • MED

    mediator complex subunit

  • MP

    MONOPTEROS

  • NPH

    NONPHOTOTROPIC HYPOCOTYL

  • PFT

    PHYTOCHROME AND FLOWERING TIME

  • PID

    PINOID

  • PIN

    PIN-FORMED

  • RT–PCR

    reverse transcription–PCR

  • SAM

    shoot apical meristem

  • SSM

    signature sequence motif

  • TRAP

    thyroid hormone receptor-associated protein

  • UTR

    untranslated region

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