MACCHI-BOU 2 Is Required for Early Embryo Patterning and Cotyledon Organogenesis in Arabidopsis
Running Title: MAB2 is required for correct embryo patterning
Corresponding Author: Masahiko Furutani
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0101, Japan
Telephone: +81-743-72-5487
Fax: +81-743-72-5489
E-mail: ma-furut@bs.naist.jp
Subject Areas: (1) growth and development
Number of black figures: 3
Number of color figures: 4
Number of table: 1
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Plant and Cell Physiology Advance Access published January 20, 2011 at Biomedische Bibliotheek on January 28, 2011
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MACCHI-BOU 2Is Required for Early Embryo Patterning and Cotyledon Organogenesis in Arabidopsis
Jun Ito, Takako Sono, Masao Tasaka and Masahiko Furutani
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
Abbreviations:AD, DNA activation domain; ARF, AUXIN RESPONSE F ACTOR; Aux/IAA, Auxin/INDOLE ACETIC ACID; BD, DNA biding domain; BDL,
BODENLOS; CCT, CENTER CITY; CDK, cyclin-dependent kinase; CycC, cyclin C; EMS, ethylmethanesulfonate; 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 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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. By 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 MAB2plays a crucial role in embryo patterning and cotyledon organogenesis, possibly through modulating expression of specific genes such as auxin-responsive genes.
Keywords: Arabidopsis thaliana, auxin response, cotyledon, embryogenesis, MED13 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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 (Blilou et al. 2005, Friml et al. 2003, Vieten et al. 2005). At least four PIN genes, PIN1, PIN3,PIN4and 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 apical-basal body axis are observed in embryos carrying multiple mutations (Blilou et al. 2005, Friml et al. 2003).
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 (Busch et al. 1996, Geldner et al. 2003, Mayer et al. 1993, Shevell et al. at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
1994). 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 F ACTOR(ARF) genes such as MONOPTEROS (MP)/ARF5 that direct the initiation of cotyledon primordia (Hay et al. 2004, Jenik and Barton 2005, Reinhardt et al. 2003). Arabidopsis possesses 23 ARF proteins that can
bind to the auxin-responsive cis-acting elements of their target genes. Auxin/INDOLE ACETIC ACID (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, 2003, 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 Chapman and Estelle 2009, Guilfoyle and Hagen 2007). 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, Hamman et al. 1999, 2002, Hardtke et al. 2004, Przemeck et al. 1996, Weijers et al. 2005, 2006). However, it is still possible that other members of these protein families contribute to cotyledon at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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 (Furutani et al. 2007, Treml et al., 2005). 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 (Benjamins et al. 2001, Bennett et al. 1995, Christensen et al. 2000), whereas mab mutants completely lack cotyledons on a pid background (Furutani et al. 2007). Recently we succeeded in identifying and characterizing the MAB4gene, which encodes a novel NPH3-like protein. MAB4is identical to ENHANCER of PINOID and its function is required for control of PIN1
polarity (Furutani et al. 2007, Treml et al. 2005).
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 MAB2is 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. at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
Results
mab2-1 is a new pid enhancer mutant
To investigate the mechanism of cotyledon organogenesis, we screened ethylmethanesulfonate (EMS)-mutagenized pid-2lines 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 to wild type (Fig. 1A, B) (Bennett et al. 1995), mab2-1 pid-2 double mutants completely lacked cotyledons (Fig. 1C).
To investigate the MAB2function, mab2-1single mutants were isolated and an analysis was made of mab2-1phenotypes. The mab2-1mutation caused aberrant cotyledon development and embryo lethality. As homozygous mab2-1plants 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 mab2-1mutants 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).
The mab2-1mutants displayed various defects in post-embryonic development. at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
Although some seedlings died before bolting, the remainder continued to develop after germination. These plants had a strongly 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 mab2-1embryos 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. at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
Expression of PIN1:GFP and DR5rev::EGFP 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, 2007), suggesting that MAB2 is involved in auxin-regulated organogenesis. To investigate the relationship between MAB2and auxin action, we analyzed PIN1:GFP expression in mab2-1 embryos to determine the effect of the mutation on auxin transport. Although mab2-1mutants had altered cotyledon numbers, expression of PIN1:GFP in the cotyledon primordia was similar to 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 mab2-1embryos, 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).
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 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
vascular bundles are arranged (Fig. 4A-C). By contrast, seven-day-old mab2-1 seedlings possessed aberrant cotyledons with an incomplete venation patterning (Fig. 4D-F); however, their hypocotyl and primary root formation, including vasculature, was fairly normal (Fig. 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 mab2-1mutation 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. S1C, D). 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 mab2-1and 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 mpT370seedlings failed to form cotyledons and to show differentiation of aligned vascular cells (Supplementary Fig. S2D-F). The basal part of at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
the mab2-1 mpT370mutant 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.
Identification of the MAB2 gene
We isolated the MAB2gene using map-based cloning. The MAB2locus 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 mab2-1mutant, a G-to-A nucleotide transition at amino acid position 1833 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 (RT)-PCR analysis showed that expression level of At1g55325was not changed in the mab2-1mutant seedlings compared to that of the wild type (Supplementary Fig. S4B). To confirm that the MAB2 gene is identical to At1g55325, we introduced At1g55325cDNA, 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 mab2-1mutation; these plants produced normal flowers without a leaf-like structure at the base of pedicel and also showed partially restored embryo development (Supplementary Fig. S3A).
We obtained other mab2alleles, mab2-2/gct-5, mab2-3, and mab2-4that carry a T-DNA insertion in At1g55325 (Fig. 5A) (Alonso et al. 2003). These mutants displayed at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
the same phenotypes as mab2-1, including aberrant cotyledon development, ectopic leaf-like formation at the base of 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-3mutant, 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 lead us to conclude that MAB2 corresponds to At1g55325.
MAB2 is a homolog of the Mediator Complex Subunit 13
MAB2 encodes a protein of 1877 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(B?ckstr?m 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 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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 (Gillmor et al. 2010, Wang and Chen 2004). 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 HIS3reporter gene activity assay, we found that MAB2 interacted with only 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 as the interaction between positive controls.
Expression patterns of MAB2
The tissue specificity of MAB2expression was examined by quantitative RT-PCR analysis. As shown in Fig. 7A, MAB2 mRNA was detected in all tissues examined. This at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
result is consistent with other published microarray data (Winter et al. 2007, Zimmermann et al. 2004). 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).
Discussion
In this study, we used a pid enhancer screen to identify a novel gene, MAB2, which is involved in cotyledon organogenesis. MAB2encodes 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: (1) cell division patterning from the onset of the 4- to 8-cell stage, presumably resulting in embryo lethality as shown in Fig. 2H; and (2) development of cotyledon primordia from the globular to heart stage (Fig. 2I, J). These defects open the possibility of correlation between MAB2function and auxin action. Auxin is a prerequisite for the establishment of the correct embryo patterning (Benková at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
et al. 2003, Blilou et al. 2005, Friml et al. 2003, 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 (Bowman and Floyd 2008, Jenik et al. 2007, Reinhardt et al. 2003). It is noteworthy that abnormal cell division at the boundary region between embryo and suspensor in mab2 mutant was also observed in embryos of auxin-response mutants such as mp(Fig. 2, Bennett et al. 1995). Additionally, in mab2embryos, expression of the auxin responsive marker DR5rev::GFP was impaired at 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 MAB2acts at the molecular level is still not understood. The synergistic effects in the mab2-1 bdl double mutant seedlings indicate that the MAB2and BDL genes function in the same process 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 AtHB8and AtHB20that are associated with procambial development (Hardtke and Berleth 1998, Mattson et al. 2003). Mutation of the MP gene results in similar seedling phenotypes as the gain-of-function bdl mutant (Hamann et al. 2002), and the mab2-1 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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 the MAB2might share its role in embryo patterning with MP, NPH4and BDL, which regulate auxin distribution and signaling. However, phenotypes of mab2mutants 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 (Fig. 1, 2, Supplementary Fig. S2). 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 the embryonic root formation.
Moreover, broad expression pattern of MAB2 mRNA in embryos implies the other 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 of the peripheral identity in the developing embryo (Kerstetter et al. 2001). In gct mutant embryos, developmental delay in embryo patterning and disordered cell patterning were observed as in mab2embryos. Gillmor et al. (2010) at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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, especially 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 MAB2encodes an Arabidopsis ortholog of MED13and 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, CDK8 subcomplex (Andrau et al. 2006, Borggrefe et al. 2002, Taatjes et al. 2004). 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 (Andrau et al. 2006, Carrera et al. 2008, Donner et al. 2007, Larschan and Winston 2005, Zhu et al. 2006), although the molecular mechanism of this process remains unclear. Homologs of Mediator and the CDK8 subcomplex show evolutionary conservation in eukaryotes including Arabidopsis (B?ckstr?m 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 showed a physical interaction at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
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 flower, and sterility. Expression analysis by quantitative RT-PCR showed that MAB2 was highly expressed in the aerial organs. Interestingly, mutation of MAB2affected 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(Clayton et al. 2008, Hong et al. 2005, Janody et al. 2003, Loncle et al. 2007, Rau et al. 2006, Treisman 2001, Yoda et al. 2005). 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 MED14and involved in the regulation of cell proliferation at the meristem (Autran et al. 2002, at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
B?ckstr?m 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 MED25subunit by B?ckstr?m 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 MAB2affected the flowering time like pft1and med8. This observation opens 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.
Material and Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotypes Landsberg erecta (L er) and Columbia (Col-0) were used as wild types. The mutant alleles used in this study were pid-2 (L er) (Christensen et al. 2000), pid-3 (Col) (Bennett et al. 1995), bdl (L er) (Hamann et al. 1999) and mpT370 (L er) (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 at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from
(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 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, Tokyo, Japan). For confocal microscopy, dissected embryos were mounted in 7% glucose.
Mapping and cloning of the MAB2 gene
The mab2-1mutant was crossed with pid-3/+or Col. Among the F2populations, seedlings without cotyledons or plants with flowers subtended by a leaf-like structure were used for map-based cloning. MAB2locus 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 coding region and the 356 bp of 3’UTR were cloned into pGWB1 (Nakagawa et al. 2007). This construct was transformed into Agrobacterium tumefaciens strain at Biomedische Bibliotheek on January 28, https://www.wendangku.net/doc/b73884819.html, Downloaded from