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First published online October 27, 2006; 10.1104/pp.106.089805 Plant Physiology 143:447-460 (2007) © 2007 American Society of Plant Biologists MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[W]Department of Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, D–50829 Cologne, Germany
The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKCc), whereas the other four remain largely unexplored. Here, we report on five members of the so-called M  or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development.
MADS-domain transcription factors play key roles in the development of higher eukaryotes. Their characteristic feature is the N-terminal MADS-domain, which is responsible for DNA binding and is highly conserved among fungi, animals, and plants. As homo- or heterodimeric complexes, and in some cases as higher-order complexes, MADS-proteins regulate gene expression by binding to CArG-box motifs in promoter regions (Egea-Cortines et al., 1999  ; Honma and Goto, 2001; Theissen and Saedler, 2001). The so-called serum response element (SRE)-type CArG-box, CC(A/T)6GG, is the DNA motif preferred by most MADS-protein complexes investigated to date (Hayes et al., 1988; Riechmann et al., 1996; de Folter and Angenent, 2006). However, some MADS-protein complexes, such as mammalian myocyte enhancer factor-2A, preferentially bind the related MEF2- or N10-type CArG-box. This motif has as general consensus C(A/T)8G but is usually more strictly defined as CTA(A/T)4TAG (Pollock and Treisman, 1991; Shore and Sharrocks, 1995). The embryo-specific AGAMOUS-LIKE 15 (AGL15) protein from Arabidopsis (Arabidopsis thaliana) and SQUAMOSA from Antirrhinum majus both have a broader spectrum of binding sites, recognizing SRE-, MEF2-, and intermediate motifs (West et al., 1998; Tang and Perry, 2003). In spite of the rather common occurrence of CArG-boxes in promoters, MADS-protein complexes are quite specific in recognizing their target promoters. Although different homodimeric MADS-protein complexes have been crystallized together with their preferred binding motif (e.g. Santelli and Richmond, 2000), the structural basis of these differences in binding specificity is still not fully understood.
In contrast to the situation in animals and fungi, the MADS-family has undergone a spectacular expansion during the evolution of plants. An initial expansion is already apparent in gymnosperms (Theissen et al., 2000
Two major monophyletic lineages have been defined within the Arabidopsis MADS-family (Alvarez-Buylla et al., 2000b
The Arabidopsis M
While the AGL67 gene is expressed in embryos and is a candidate for regulating aspects of late embryo development (de Folter et al., 2004 In a first attempt to functionally characterize the AtMIKC* proteins, we performed protein-interaction and DNA-binding studies for the five pollen-expressed members of this subgroup. We show that between members of the two monophyletic AtMIKC* lineages, five heterodimeric protein complexes can be formed, at least four of which exist in planta. They preferentially bind MEF2-type CArG-boxes in vitro, and these preferred binding motifs are strongly overrepresented in the proximal region of promoters that are activated during the last stages of pollen development, at the time of AtMIKC* expression. Our results suggest that the AtMIKC* protein complexes play an important role in the regulation of transcription during late pollen development, and this is confirmed by a preliminary functional analysis of mutant pollen in which all five AtMIKC* complexes are either absent or strongly reduced in abundance.
Cloning of AtMIKC* cDNAs from Pollen
We cloned the full-length cDNAs of five AtMIKC* genes (AGL30, AGL65, AGL66, AGL94, and AGL104) from a cDNA pool obtained from mature Arabidopsis pollen grains, using gene-specific primers. AGL30 had previously been regarded a pseudogene based on the cloning of a truncated splice variant with a stop codon in the fifth exon (Kofuji et al., 2003
Given that MADS-proteins generally function in dimeric complexes, we tested the interactions between AGL30, AGL65, AGL66, AGL94, and AGL104 in the yeast two-hybrid (Y2H) system. All bait constructs showed autoactivation in yeast, which was abolished after deletion of a C-terminal fragment (
To verify AtMIKC* protein interactions in planta, bimolecular fluorescence complementation (BiFC) was performed using the full-length open reading frame (ORF) sequences (Fig. 1B). The AGL30/66, AGL65/66, and AGL65/104 complexes, which we demonstrated in yeast, could be detected in the nuclei of transiently transformed tobacco (Nicotiana benthamiana) leaf cells (Fig. 1C), indicating that no pollen-specific factors are required for their targeting to the nucleus. The fluorescence signal colocalized with the 4',6-diamidino-2-phenylindole nuclear stain and had the exact spectral properties of the yellow fluorescent protein (YFP; data not shown). We were unable to confirm interaction between AGL66 and AGL94 using this technique, even though these proteins strongly associated in yeast. On the other hand, interaction between AGL30 and AGL104, which was not evident in yeast, could be reliably detected in planta (Fig. 1D). In agreement with our Y2H results, no interaction could be observed between other AtMIKC* protein combinations. All interactions between AtMIKC* proteins are summarized in Figure 1E.
To test their ability to bind DNA, the five pollen-expressed AtMIKC* proteins were synthesized in vitro in a cell-free system and used in electrophoretic mobility shift assay (EMSA) experiments. The five protein combinations capable of physical interaction (namely AGL30/66, AGL65/66, AGL94/66, AGL30/104, and AGL65/104; Fig. 1E) showed appreciable binding to a randomized mixture of SRE- and MEF2-type CArG-boxes (Fig. 1F). No DNA interaction could be observed for other AtMIKC* protein combinations (Fig. 1F) nor for any of the proteins alone (data not shown). The AGL30/66 and AGL65/66 protein complexes, which exhibited the strongest DNA binding in this experiment, were chosen for a random binding site selection (RBSS) experiment to determine their preferred binding motifs. After five iterative rounds of RBSS, both complexes showed a very pronounced preference for the MEF2-type CArG-box with consensus CTA(A/T)4TAG (Fig. 2A ). This motif accounted for 78% and 63% of all sequences enriched by the AGL30/66 and AGL65/66 complexes, respectively (87 out of 111 and 83 out of 131 sequences). One particular motif, CTA(TTTT)TAG/CTA(AAAA)TAG, was preferred most strongly by both complexes. The second most preferred motif was CTA(TATA)TAG, in the case of AGL30/66, and the N9-type CArG-like motif CTA(TTT)TAG/CTA(AAA)TAG for AGL65/66. Only 1% of the sequences was of the SRE type [CC(A/T)6GG], indicating that this motif is not preferred by AtMIKC* complexes. The complete RBSS datasets are listed in Supplemental Figure S2.
We corroborated our RBSS results with competitive EMSA experiments, confirming the strong preference of the AGL30/66 and AGL65/66 complexes for MEF2 motifs and in particular for the CTA(TTTT)TAG/CTA(AAAA)TAG motif (Fig. 3 ). We also used the competitive EMSA approach to investigate the DNA-binding preference of the three other AtMIKC* protein complexes. The AGL30/104 complex showed a similar behavior as AGL30/66, clearly preferring the MEF2-type CArG-box motifs CTA(TTTT)TAG and CTA(TATA)TAG to the N9-type motif CTA(TTT)TAG and the SRE-type motif CCTATTTAGG (Fig. 3). For the AGL94/66 complex, accurate quantification of DNA binding was impossible, because it produced very diffuse shifted bands in our hands and also for AGL65/104, which in general bound very weakly to DNA in vitro (Fig. 1F).
MEF2 Motifs Are Specifically Overrepresented in Late Pollen-Specific Promoters
The AtMIKC* genes are highest expressed during the tricellular and mature stages of pollen development (Honys and Twell, 2004
To address this matter, we reanalyzed the pollen transcriptome dataset from Honys and Twell (2004)
Subsequently, we screened the 3-kb upstream regions of these genes for the presence of MEF2-type CArG-boxes, and found them in 31 BCP-specific and in 152 TCP/MPG-specific promoters (Fig. 2B). Remarkably, 22.9% of the TCP/MPG-specific promoters contain a MEF2 motif, while this is only the case for 13.1% of all Arabidopsis promoters genome wide and 9.9% of the BCP-specific promoters (Fig. 2B). In contrast, SRE-type CArG-boxes, which are not bound in vitro by the AtMIKC* complexes (Fig. 2A), are overall not more abundant in BCP- and TCP/MPG-specific promoters (17.2% and 19.0%, respectively) than in all promoters genome wide, where they occur in 18.8% of all promoters (Supplemental Table S2). This percentage is in agreement with the report from de Folter and Angenent (2006)
Because AtMIKC* protein complexes are able to discriminate between the 16 different MEF2 motifs in vitro (Fig. 2A), we investigated whether these motifs occur with comparable frequencies in Arabidopsis promoters, or whether some of them occur more frequently than the rest. We first screened the 3-kb upstream regions of all Arabidopsis genes for the presence of MEF2-type CArG-boxes using the Patmatch tool on The Arabidopsis Information Resource (TAIR) Web site (www.arabidopsis.org) and identified 4,571 such motifs in 4,121 different promoters (Fig. 2B). For our study, we defined a promoter as the entire region upstream of the ATG start codon, including the 5' untranslated region (5'UTR). We found that the CTA(TTTT)TAG/CTA(AAAA)TAG and CTA(TTTA)TAG/CTA(TAAA)TAG motifs each account for over 16% of all MEF2 motifs in 3-kb promoters, whereas the palindromic CTA(TTAA)TAG motif only represents 2% of the cases (Fig. 2B). Hence, some of the MEF2 motifs occur very frequently in Arabidopsis promoters, while others are clearly underrepresented.
Next, we repeated this analysis with all BCP- and TCP/MPG-specific promoters and found that the relative occurrence of the 16 different MEF2 motifs in these promoters differs from that in all promoters genome wide. In particular, the CTA(TTTT)TAG/CTA(AAAA)TAG motif, which is the most preferred binding motif of the AtMIKC* complexes in vitro (Fig. 2A), is strongly overrepresented in TCP/MPG-specific promoters (27.9% compared to 16.9% in all Arabidopsis promoters; Fig. 2B). None of the other MEF2 motifs shows this tendency. The palindromic CTA(TATA)TAG motif, bound quite well in vitro by the AGL30/66 complex (Fig. 2A), is underrepresented; it accounts for just 6.7% of the motifs in TCP/MPG-specific promoters, compared to 13.4% in all 3-kb promoter regions genome wide and 13.5% in BCP-specific promoters. The limited occurrence of this motif in TCP/MPG-specific (but not in BCP-specific) promoters might be related to the function of AGL18 in mature pollen. Because AGL18 is closely homologous to AGL15 (Alvarez-Buylla et al., 2000a
To allow comparison, we also screened all Arabidopsis 3-kb promoters for N9- and SRE-type CArG-boxes (Supplemental Table S2). The relative occurrence of the 64 possible SRE motifs is also nonrandom, with the CCTTTTTTGG/CCAAAAAAGG and CCATTTTTGG/CCAAAAATGG motifs accounting for over 8% of all SRE motifs in Arabidopsis promoter regions, while several other SRE motifs represent less than 1% of the total number. In BCP-specific, but not in TCP/MPG-specific, promoters, one particular motif (CCTTTTTTGG/CCAAAAAAGG) is strongly overrepresented (accounting for 17.5% of all SRE motifs in BCP-specific promoters and for only 8.1% in TCP/MPG-specific promoters and 8.9% in all Arabidopsis promoters; Supplemental Table S2), suggesting that it could be the preferred binding site for a non-AtMIKC* MADS-protein complex functioning during the BCP and/or TCP stage of pollen development. AGL18 would, again, be a good candidate for being part of such a complex, based on its expression profile throughout pollen development and the fact that the related AGL15 protein binds well to SRE motifs (Tang and Perry, 2003 Whether the AtMIKC* complexes preferentially bind one of the two complements of a nonpalindromic MEF2 motif or whether they bind both complements to the same extent is a question that remained unanswered after our RBSS experiments, which are essentially nondirectional in nature. Our in silico analyses showed that the two complements of nearly all MEF2 motifs occur equally often in all promoters genome wide (Fig. 2C). In TCP/MPG-specific promoters, the CTA(TTTT)TAG and CTA(AAAA)TAG complements also occur with similar frequencies (26 and 24 times, respectively), indicating that the directionality of this motif in a promoter plays no role in its recognition by the AtMIKC* protein complexes in vivo. However, the complements of two other motifs, CTA(TATT)TAG/CTA(AATA)TAG and CTA(TAAT)TAG/CTA(ATTA)TAG, are found in quite unequal proportions in TCP/MPG-specific promoters (2.2% versus 8.4% and 1.7% versus 8.4%, respectively; Fig. 2C), suggesting that the AtMIKC* complexes might have a different affinity for binding to the two complements of these motifs. Both motifs are moderately represented in the RBSS dataset for the AGL65/66 complex (Fig. 2A).
Since spatial positioning of cis-acting elements in a promoter is often an important factor in the regulation of gene expression, we investigated the spatial distribution of CArG-boxes in Arabidopsis 3-kb upstream regions. We found that MEF2, SRE, and N9-type motifs are distributed quite homogeneously across the promoters genome wide (Fig. 4 ). For example, 50% of all MEF2 motifs are found within 1,200 bp upstream of the ATG and 27% are positioned in the most proximal 500 bp of a promoter (Supplemental Table S2). In BCP-specific promoters, the spatial distribution of MEF2 motifs is comparable to that in the promoters genome wide. In contrast, 51% of the MEF2 motifs in TCP/MPG-specific promoters are located within 500 bp upstream of the ATG, and for the CTA(TTTT)TAG/CTA(AAAA)TAG motif, this is even 60% (Fig. 4; Supplemental Table S2). SRE motifs, in contrast, tend to have a proximal positioning in BCP-specific, but not in TCP/MPG-specific, promoters.
Loss of AtMIKC* Complexes Affects Pollen Germination in Vitro We identified T-DNA insertion lines for four of the AtMIKC* genes (as described in "Materials and Methods") and obtained homozygous plants for each line. We then combined the different mutant alleles by crossing, ultimately resulting in the following single and double mutants: agl65, agl66, agl94, agl104, agl65/66, agl65/104, and agl66/104. Only for the agl104 mutant residual expression of the full-length transcript could be observed (Fig. 6; consistently less than 30% of the wild-type level), while the transcripts of the other AtMIKC* genes were expressed as truncated forms and/or were completely absent in their respective mutants (see "Materials and Methods"). Consistent with the male gametophyte-specific expression of the AtMIKC* genes, no sporophytic phenotype could be observed for any of the mutant lines. All mutant plants also exhibited a normal fertility.
Assuming that the late pollen-expressed AtMIKC* complexes might be important for pollen germination, we performed in vitro germination assays with pollen grains from our different single and double mutant Arabidopsis lines lacking one or more functional AtMIKC* complexes. In Figure 5A , the different genotypes are listed, together with the AtMIKC* complexes still present in each mutant. Pollen viability, as scored with fluorescein-3',6'-diacetate, was unaffected in all mutants and always higher than 90% (data not shown). Each of the AtMIKC* complexes contains either the AGL66 or AGL104 protein (Fig. 1E), and the loss of functional AGL66 (in the agl66 mutant) or the strong reduction in AGL104 abundance (in the agl104 mutant) does not affect pollen germination in vitro (Fig. 5B). This indicates that the complexes containing AGL66 are completely functionally redundant with those containing AGL104. On the other hand, loss of functional AGL65 protein (in the agl65 mutant) results in a significant reduction of pollen germination efficiency (Fig. 5B). This demonstrates that the remaining three complexes (AGL30/66, AGL94/66, and AGL30/104) are unable to completely compensate for the loss of AGL65/66 and AGL65/104 in this mutant. The AGL65/66 and AGL65/104 complexes are therefore most likely functionally redundant with each other but not with the other three AtMIKC* complexes.
The additional loss of two other complexes (AGL30/66 and AGL94/66) in the agl65/66 double mutant only slightly reduces the germination efficiency further compared to the agl65 mutant, as does the additional reduction of AGL30/104 complex abundance in the agl65/104 double mutant (Fig. 5B). This observation indicates that the AGL30/66 and AGL94/66 complexes are largely functionally redundant with the AGL30/104 complex and that they are able to sustain around 40% of the wild-type pollen germination efficiency in vitro. When all five AtMIKC* complexes are affected (either functionally lost or strongly reduced in abundance, in the agl66/104 double mutant), pollen grains are virtually unable to germinate in vitro (Fig. 5B). This confirms the essential role of the AtMIKC* complexes in late pollen development and pollen tube growth.
The 152 TCP/MPG-specific genes with a MEF2 motif in their 3-kb promoter (listed in Supplemental Table S2), whose expression is up-regulated following the appearance of the AtMIKC* complexes during the TCP stage, are potential direct targets of the AtMIKC* transcription factor complexes. Among them are various genes with a function related to vesicle transport and cytoskeleton, cell wall, and signal transduction (Supplemental Fig. S3). These classes are essential for pollen germination and are generally overrepresented in the transcriptome of mature pollen (Honys and Twell, 2003
MADS-box genes are key regulators of a range of higher plant-specific developmental programs. However, our knowledge of the MADS family in Arabidopsis is mainly limited to the MIKCc subgroup, as only two non-MIKCc genes have been functionally characterized to date (namely AGL37 and AGL80, both from the M  subgroup; Köhler et al., 2003; Portereiko et al., 2006). In this study, we initially characterized five additional non-MIKCc MADS-box genes, all belonging to the M or AtMIKC* subgroup and specifically expressed during the tricellular and mature stages of pollen development (Honys and Twell, 2004). We found that five heterodimeric complexes were formed between these AtMIKC* proteins in yeast, at least four of which also exist in planta (Fig. 1, A–D). The AGL30/104 complex could be observed by EMSA and BiFC, but not in yeast. This apparent discrepancy might be caused by the weakness of their interaction, because complementation of YFP (in BiFC) has been reported to stabilize transient and weak protein interactions (Walter et al., 2004), and interaction with DNA (in EMSA) can have a comparable effect. Conversely, we could demonstrate an interaction between AGL94 and AGL66 in yeast but not in planta. In EMSA experiments, the AGL94-AGL66 protein combination consistently produced diffuse shifted bands (Fig. 1F), indicating suboptimal DNA binding. Taken together, these observations indicate that the AGL94/66 complex might not exist in vivo, but because it does bind DNA in vitro, we are currently unable to fully exclude a role for AGL94/66 in Arabidopsis pollen. Interestingly, interactions exclusively occur between members of the two monophyletic lineages within the AtMIKC* subgroup (AGL30, AGL65, and AGL94 on one hand, and AGL66 and AGL104 on the other hand) and never between members of the same lineage (Fig. 1E). This indicates an ancestral scenario in which only one heterodimeric AtMIKC* complex existed, formed between an AGL65-like and an AGL66-like protein. Gene duplication events have subsequently led to the expansion of both MIKC* lineages and resulted in the five AtMIKC* complexes present in extant Arabidopsis pollen.
Our experiments suggest a high degree of functional redundancy between the five AtMIKC* complexes. The AGL30/66 and AGL65/66 complexes, which can be considered representative for all five AtMIKC* complexes, most avidly bind the same MEF2 motif CTA(TTTT)TAG/CTA(AAAA)TAG and show hardly any affinity for SRE motifs (Fig. 2A), which are preferred by most reported MADS-proteins (de Folter and Angenent, 2006 Our in vitro pollen germination experiments (Fig. 5) further clarified the redundancy between the different complexes. They revealed that the AGL65/66 and AGL65/104 complexes are functionally redundant with each other, but not with the other three complexes, and that AGL30/66 and AGL94/66 are redundant with AGL30/104. Our in vitro observations based on DNA-binding preferences (suggesting redundancy between AGL30/66 and AGL30/104, but not between AGL30/66 and AGL65/66) are therefore in good agreement with the in vivo situation. The functional interchangeability of the AGL66 and AGL104 proteins is actually not that surprising, considering the high homology they share at the protein level. Their MADS and I domains are nearly identical in sequence (Supplemental Fig. S1), indicating that the AGL66 and AGL104 genes originated from a relatively recent gene duplication event.
MADS complexes have always been found to bind a relatively broad spectrum of DNA motifs. An in-depth analysis of the occurrence of their binding motifs in the genome therefore seemed irrelevant. Only the AGL15 homodimer has been reported to exhibit preference for one particular MEF2 motif, CTA(TATA)TAG, but it also binds SRE- and N9-type motifs with comparable affinity (Tang and Perry, 2003
We then carried out a large-scale in silico analysis in which we examined the overall and relative abundance of the individual CArG-boxes, as well as their spatial positioning in Arabidopsis promoters genome wide and in pollen-specific promoters. Thereby, we took advantage of a unique single-cell transcriptome dataset covering the different stages of pollen development (Honys and Twell, 2004
Our example illustrates that specific binding sites can be overrepresented in promoters that share a similar, narrow expression pattern, and this reflects the binding specificity of transcription factors functioning in that specific cell type or during a predefined developmental stage. We think this in silico approach is in general a powerful, indirect tool to help uncover new transcriptional regulatory networks in specific cell types or developmental stages. The concept of using experimentally well-defined transcription factor binding sites as a starting point for a large-scale genomics and single cell-type transcriptomics in silico analysis also seems feasible for other cell types than pollen, and for any transcription factor with a clearly defined DNA-binding preference. The main difficulty lies in obtaining transcriptome data that allow such in silico analyses, but recent technical advances have enabled the purification of trichomes (Zhang and Oppenheimer, 2004
With our in silico approach, we identified 152 putative direct target genes of the AtMIKC* complexes in Arabidopsis pollen. These genes are specifically expressed during the last two stages of pollen development and contain at least one MEF2 motif in their 3-kb promoter (Supplemental Table S2). We tested the expression of a random selection of these genes by RT-PCR and found that the majority of them are affected in agl66/104 double mutant pollen (Fig. 6), implying that these genes are truly downstream of the AtMIKC* complexes, and that our in silico approach is reliable for predicting target genes of transcription factors. Two of the most strongly affected genes (At2g44560 and At5g19610) contain the CTA(TAAA)TAG and CTA(AATA)TAG motifs in their proximal promoter, respectively. In vitro, these motifs are both bound rather well by the AGL30/66 and/or AGL65/66 complexes (Fig. 2A), but they are not overrepresented in TCP/MPG-specific promoters (Fig. 2B). This observation raises the possibility that MEF2 motifs other than the most preferred and overrepresented CTA(TTTT)TAG/CTA(AAAA)TAG motif can be bound by the different AtMIKC* complexes in vivo, or, alternatively, that these affected genes are actually indirect targets rather than direct targets. We are currently unable to exclude either possibility, but chromatin immunoprecipitation experiments could help to further clarify this matter. In addition, the expression of some of the in silico predicted direct target genes with proximal CTA(TTTT)TAG/CTA(AAAA)TAG motif in their promoter is unaffected in agl66/104 double mutant pollen (Fig. 6), suggesting that the presence of AtMIKC* binding sites in the proximal promoter is not always sufficient to make a late pollen-specific gene a direct target of the AtMIKC* complexes. How MADS-protein complexes achieve their high degree of target specificity and how they distinguish the CArG-boxes in their target promoters from all other CArG-boxes in the genome are important questions in plant development. Current hypotheses assume the involvement of accessory DNA-binding factors associating with MADS-protein complexes or a role for the nucleotides flanking CArG-boxes in promoters (de Folter and Angenent, 2006 One of the genes demonstrated to be down-regulated in agl66/104 double mutant pollen is MYB97, one of the seven transcription factor-encoding genes among the predicted direct targets (Fig. 6; Supplemental Fig. S3). The fact that other transcription factors are downstream of the AtMIKC* complexes highlights their importance in transcriptional regulation during late pollen development. It suggests that the AtMIKC* complexes rank high in the hierarchy of a pollen-specific transcriptional network and that many more BCP- and TCP/MPG-specific genes are likely under indirect control by the AtMIKC* complexes. The profound effect of the virtual loss of all AtMIKC* complexes on pollen germination efficiency in vitro (Fig. 5) clearly illustrates the pivotal role these complexes play in pollen.
The underlying cause of the pollen germination defect remains to be elucidated, but it is likely the result of multiple factors, because quite a few of the putative AtMIKC* target genes have reported or proposed functions in pollen germination. For example, seven cation/proton exchanger-encoding genes are among the putative direct targets (Supplemental Fig. S3), and at least two of them (CHX8 and CHX24) are indeed differentially expressed in agl66/104 double mutant pollen (Fig. 6). These proteins are postulated to allow osmotic adjustment and ion homeostasis during pollen desiccation, rehydration, and germination (Sze et al., 2004 Based on the severity of the in vitro pollen germination phenotype of the agl66/104 double mutant (Fig. 5), one might expect this mutant to be male sterile. Intriguingly, however, it shows no impaired fertility, making it unlikely that the AtMIKC* complexes play a role in determining pollen fertility itself. The mutant phenotype might therefore be more subtle in vivo, perhaps affecting the speed and efficiency of pollen germination and tube growth. A more detailed molecular and functional characterization of Arabidopsis mutants lacking the different AtMIKC* complexes should further clarify the exact biological role of these atypical MADS-box genes in pollen development.
Isolation and Cloning of MIKC* cDNAs
Mature pollen grains were isolated from open flowers of the Arabidopsis (Arabidopsis thaliana) Columbia accession (grown in a growth chamber at 22°C, with 16 h of light at around 140 µmol m–2 s–1), using the protocol of Honys and Twell (2003)
The full-length ORF sequences of the five pollen-expressed AtMIKC* genes were cloned into the pGADT7 prey vector and the pGBKT7 bait vector (CLONTECH), which carry the LEU2 and TRP1 selection markers, respectively (AGL30 with NcoI and BamHI, AGL65 with NdeI and BamHI, AGL66 with NcoI, AGL94 with EcoRI and BamHI, and AGL104 with XmaI). The ORF of the pollen-expressed MIKCc gene AGL18 was also cloned into both vectors (with NcoI). Yeast (Saccharomyces cerevisiae) strain AH109 (CLONTECH) was sequentially transformed with all available combinations of bait and prey constructs. The transformation mixtures were plated out onto synthetic dropout medium lacking Leu and Trp to test for transformation efficiency and onto synthetic dropout medium lacking Leu, Trp, and Ade to test for protein-protein interactions. For all AtMIKC* proteins, a strong autoactivation of the bait constructs was observed, and to circumvent this problem, a C-terminal part was removed (by restriction digestion with PstI in case of AGL94 and by PCR with nested primers and recloning in case of the other genes), resulting in a series of
We used the BiFC system described by Bracha-Drori et al. (2004)
The AtMIKC* ORFs were cloned into the pSPUTK vector (Promega) using NcoI (AGL30 and AGL66), SalI and EcoRI (AGL94), HindIII and BamHI (AGL65), and SmaI (AGL104). These constructs (500 ng of plasmid) were used for coupled in vitro transcription and translation with the TnT SP6-Coupled Reticulocyte Lysate system (Promega). Radioactively labeled Met ([35S]Met) was included in this reaction to allow detection of the synthesized proteins on SDS-PAGE gel. In samples meant for testing the DNA-binding properties of heterodimeric complexes, two proteins were synthesized together in one reaction. As a control PpMADS2, a homodimerizing MIKC* protein from the moss Physcomitrella patens was included (R. Hallinger, W. Verelst, W. Faigl, H. Saedler, and T. Münster, unpublished data). In an EMSA reaction, 2 µL of protein sample was mixed with 1 µL of DNA probe in a reaction mixture containing 2.5% (w/v) CHAPS, 9 mM HEPES, pH 7.3, 1.4 mM EDTA, pH 8.0, 8% (w/v) glycerol, 1.33 mM spermidine, 0.9 mM dithiothreitol, 75 ng/µL bovine serum albumin (New England Biolabs), and 11.5 µg/µL autoclaved calf thymus DNA (Serva) and incubated on ice for 30 min. Subsequently, protein-DNA complexes were separated from unbound probes by electrophoresis on a 4% nondenaturing polyacrylamide gel. Bands were visualized using a phosphor screen and a Typhoon 8600 phosphor imager (Molecular Dynamics). For the EMSA experiment illustrated in Figure 1F, an equimolar mixture of two randomized probes was used: GATCCTGTCGNNNCC-(A/T)6-GGNNNGAGGCGAAT (SRE-type CArG-box) and GATCCTGTCGNNNC-(A/T)8-GNNNGAGGCGAAT (relaxed MEF2-type CArG-box). DNA probes were labeled with [
For RBSS (Pollock and Treisman, 1990 In competitive EMSA experiments, the only difference with the regular EMSA approach was that the proteins were incubated on ice with unlabeled double-stranded 62-mer fragments for 20 min, prior to the addition of a radioactively labeled probe to the EMSA mixture, followed by another 20 min of incubation. Both the labeled and unlabeled probes were identical to the randomized probe used for RBSS but with a more defined core sequence [NNNCTA(TTTT)TAGNNN, NNNCTA(TATA)TAGNNN, NNNCTA(TTT)TAGNNNN, NNNCTA(TAAT)TAGNNN, and NNNCCTATTTAGGNNN]. These sequences were chosen based on the results of the RBSS experiments (Fig. 2A; Supplemental Fig. S2). The double-stranded competitor probes were produced by boiling a mixture of two complementary single-stranded oligonucleotides, followed by gradual cooling to room temperature and subsequent purification over columns designed for double-stranded DNA isolation (Roche). The resulting double-stranded probes were diluted to a concentration of 100 ng/µL and their concentration was verified both spectrophotometrically and on agarose gel. These cold competitor probes were generally used at an excess of 30- and 125-fold relative to the labeled probe, unless stated otherwise.
Total RNA was isolated from mature pollen (as described above), which had been harvested from wild-type and agl66/104 double mutant plants grown in a greenhouse during summer, with temperature controlled at 22°C and 16 h of light at around 120 µmol m–2 s–1. Primers for RT-PCR on predicted target genes were designed in the 3' region of the ORF, they usually coincided with probe sets for these genes used on the ATH1 microarray chip (Affymetrix), and wherever possible spanned an intron. RT-PCR was performed for 14 predicted target genes using 150 ng cDNA and gene-specific primers. As a control, 18S ribosomal RNA was amplified using the QuantumRNA primer-competimer approach from Ambion, with a 2:8 ratio of primer-competimer and 35 PCR cycles. The number of PCR cycles was optimized for each gene: 20 cycles for At1g74000, At1g17540, At4g18700, At5g35390, At3g46520, At1g13890, and At5g64790; 25 cycles for At4g26930, At5g55980, At2g28180, At5g19610, and At2g05850; 30 cycles for At2g44560, AGL66, and AGL104; and 35 cycles for At5g37060.
For in vitro germination of pollen the protocol of Li et al. (1999)
Viability staining of pollen was performed with fluorescein-3',6'-diacetate according to Eady et al. (1995)
The protein sequence alignment in Supplemental Figure S1 was created with ClustalW (Chenna et al., 2003 The sequence data from this article can be found in the GenBank data library, under accession numbers DQ446459 (AGL30), NM_101733 (AGL65), NM_106447 (AGL66), NM_105623 (AGL94), NM_102063 (AGL104), and NM_115599 (AGL18).
The following materials are available in the online version of this article.
We thank Drs. Joachim Uhrig and Klaus Richter for vectors and practical help with BiFC, Dr. Suzanne Kuijt for assistance with confocal microscopy, and Drs. Zsuzsanna Schwarz-Sommer, Rainer Birkenbihl, Malgorzata Domagalska, and two anonymous reviewers for helpful comments and critically reading the manuscript. Received September 26, 2006; accepted October 25, 2006; published October 27, 2006.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wim Verelst (verelst{at}mpiz-koeln.mpg.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.089805 * Corresponding author; e-mail verelst{at}mpiz-koeln.mpg.de; fax 49–221–5062–113.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 Alvarez-Buylla ER, Liljegren SJ, Pelaz S, Gold SE, Burgeff C, Ditta GS, Vergara-Silva F, Yanofsky MF (2000a) MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. Plant J 24: 1–11[CrossRef][ISI][Medline] Alvarez-Buylla ER, Pelaz S, Liljegren SJ, Gold SE, Burgeff C, Ditta GS, de Pouplana LR, Martinez-Castilla L, Yanofsky MF (2000b) An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proc Natl Acad Sci USA 97: 5328–5333 Becker A, Theissen G (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol Phylogenet Evol 29: 464–489[CrossRef][ISI][Medline] Becker A, Winter K-U, Meyer B, Saedler H, Theißen G (2000) MADS-box gene diversity in seed plants 300 million years ago. Mol Biol Evol 17: 1425–1434 Becker JD, Boavida LC, Carneiro J, Haury M, Feijo JA (2003) Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome. Plant Physiol 133: 713–725 Birkenbihl RP, Jach G, Saedler H, Huijser P (2005) Functional dissection of the plant-specific SBP-domain: overlap of the DNA-binding and nuclear localization domains. J Mol Biol 352: 585–596[CrossRef][ISI][Medline] Birnbaum K, Jung JW, Wang JY, Lambert GM, Hirst JA, Galbraith DW, Benfey PN (2005) Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat Methods 2: 615–619[CrossRef][ISI][Medline] Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40: 419–427[CrossRef][ISI][Medline] Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31: 3497–3500 Cheung AY, Chen CY, Glaven RH, de Graaf BHJ, Vidali L, Hepler PK, Wu H (2002) Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. Plant Cell 14: 945–962 De Bodt S, Raes J, Van de Peer Y, Theißen G (2003) And then there were many: MADS goes genomic. Trends Plant Sci 8: 475–481[CrossRef][ISI][Medline] de Folter S, Angenent GC (2006) Trans meets cis in MADS science. |
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ABSTRACT
RESULTS
enicová et al., 2003
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C), including suspected activation domains. Using these truncated bait constructs and full-length prey constructs, we found four reciprocally interacting pairs: AGL66 dimerizes with AGL30, AGL65, and AGL94, whereas AGL104 interacts only with AGL65. The interactions of AGL65 with AGL66 and with AGL104 in yeast was previously reported (de Folter et al., 2005
