* Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan
National Institute for Basic Biology, Okazaki, Japan
Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan
Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki, Japan
Correspondence: E-mail: mhasebe{at}nibb.ac.jp.
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Abstract |
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Key Words: MADS-box Arabidopsis genome gene family sporophyte gametophyte
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Introduction |
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MADS-box genes are characterized by approximately 180-bp DNA sequences, termed the MADS-box, which encodes a DNA-binding and dimerization domain, the MADS domain (reviewed in Shore and Sharrocks 1995). The MADS domain is conserved across a diverse range of organisms, from fungi, slime mold, and metazoans to land plants; in addition, putative MADS domains have been found in bacteria (Mushegian and Koonin 1996). MADS-box genes in eukaryotes other than land plants are divided into two groups, SRF-type and MEF2-type, based on phylogenetic analyses (Theissen, Kim, and Saedler 1996; Hasebe and Banks 1997), and their functions vary, even within the two groups. The SRF-type MADS-box genes are involved mainly in cell growth and differentiation in mammalian cells and mating type differentiation in budding yeast, whereas the MEF2-type genes function in muscle cell differentiation (reviewed in Theissen et al. 2000). In land plants, MADS-box genes were first identified as floral homeotic selector genes (Sommer et al. 1990; Yanofsky et al. 1990); all floral homeotic genes except one were shown to be MADS-box genes (reviewed in Weigel and Meyerowitz 1994). Two groups of land plant MADS-box genes are recognized: MIKCC- and MIKC*-type MADS-box genes (Henschel et al. 2002). MIKCC-type genes are composed of a MADS (M) domain, an intervening (I) domain, a keratin-like (K) domain, and a C-terminal (C) domain, and they have been reported from a variety of land plants, including seed plants, pteridophytes, and bryophytes (reviewed in Theissen et al. 2000; Henschel et al. 2002). MIKC*-type genes have been reported only from lower land plants, including a moss (Henschel et al. 2002) and a club moss (Svensson, Johannesson, and Engstrom 2000), which are distinguished by their different intron/exon structure in the I domain, as compared with MIKCC-type genes (Henschel et al. 2002). Another type of MADS-box genes that lack the K domain (called M-type MADS-box genes here) was recently found during the Arabidopsis Genome Sequencing project (Alvarez-Buylla et al. 2000b), but their expression patterns have not yet been studied. Alvarez-Buylla et al. (2000b) divided SRF-, MEF2-, M-, MIKCC-, and MIKC*-type MADS-box genes into two groups, type I and II, according to the phylogenetic analyses and signature amino acid residues in the MADS domain. This hypothesis should be assessed using more data, such as all the MADS-box genes in the Arabidopsis (Arabidopsis thaliana) genome.
Although the functions of MIKC*- and M-type MADS-box genes are unknown, MIKCC-type MADS-box genes have diverse functions. In addition to the floral homeotic genes, MIKCC-type genes function in floral transition (Michaels and Amasino 1999; Sheldon et al. 1999; Borner et al. 2000; Hartmann et al. 2000; Lee et al. 2000), cell differentiation of fruits (Liljegren et al. 2000; Liljegren and Yanofsky 2000; Vrebalov et al. 2002), and root architecture (Zhang and Forde 1998). In addition to the roles of MIKCC-type MADS-box genes in sporophyte (diploid) generation, some angiosperm and fern MIKCC-type MADS-box genes are expressed in gametophyte (haploid) generation. The detailed patterns of expression in fern gametophytes are unknown (Münster et al. 1997; Hasebe et al. 1998), but DEFH125 of snapdragon, ZmMADS2 of maize, and At3g57390/AGL18 of Arabidopsis are expressed in mature pollen, the male gametophyte, although their functions are unknown (Zachgo, Saedler, and Schwarz-Sommer 1997; Alvarez-Buylla et al. 2000a; Heuer, Lörz, and Dresselhaus 2000). At3g57390/AGL18 is expressed in the female gametophyte in addition to the male gametophyte, but its function has not been analyzed (Alvarez-Buylla et al. 2000a). Land plants are of monophyletic origin from a freshwater ancestor believed to be a charophycean green alga, whose plant body is gametophytic, except for a unicellular zygote (Graham, Cook, and Busse 2000), indicating that gametophytic and/or zygote-specific MADS-box genes are likely ancestral. One of the possible hypotheses for the evolution of MADS-box genes is that they were involved in unknown functions in gametophytes and/or zygotes, and were subsequently recruited for sporophyte development during the course of land plant evolution as the sporophyte came to dominate the gametophyte. The characterization of MADS-box genes expressed in gametophytes in land plants is important to the understanding of the origin and evolution of MADS-box genes.
The genome of Arabidopsis thaliana Columbia-0 has been almost entirely sequenced (The Arabidopsis Genome Initiative 2000), and it is predicted to contain 82 MADS-box genes (Riechmann 2002). Phylogenetic analyses and expression analyses of all the MADS-box genes in the Arabidopsis genome were performed to examine the evolution and diversity of plant MADS-box genes, and especially to evaluate (1) the previous classification of type I and II MADS-box genes and their evolutionary relationships, (2) whether gametophyte-specific MADS-box genes in addition to At3g57390/AGL18 exist in the Arabidopsis genome, and (3) the evolution of expression patterns in the MADS-box gene family.
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Materials and Methods |
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Total RNA for a target was extracted using an RNeasy Plant Mini Kit (QIAGEN, Valencia, Calif.) from roots collected from juvenile plants grown for 20 days after sowing on an agar plate, juvenile plants with a few young rosette leaves and roots grown for 8 days after sowing, mature rosette leaves, inflorescences containing flower buds at stages less than stage 12, flowers at stages 12 and 13, pollen, and siliques 0 to 7 days after flowering. The stages of flower development followed that devised by Smith, Bowman, and Meyerowitz (1990). The agar plates used for root cultivation contained 2.2 g/L Murashige and Skoog culture medium basal salt (Wako Pure Chemical Industries, Osaka, Japan), 1 ml/L 1,000x Gamborg's vitamin solution (Sigma-Aldrich), 1% sucrose, and 0.8% agar. Pollen was collected by vortexing flowers in TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) for 30 s, removing the flower debris, centrifuging at 10,000 x g for 1 min, and filtering through a 50-µm nylon mesh (Kyoshin Riko Inc., Tokyo, Japan). Poly-A+ RNA, purified using DynaBeads (Dynal, Oslo, Norway), was used for RNA extracted from siliques.
Total RNA was reverse-transcribed to cDNA in the presence of [32P]-dCTP (Amersham Bioscience). Five µg of total RNA or poly-A+ RNA, purified using 5 µg of total RNA, and 10 pmol of adaptor primer were mixed, heated to 70°C for 10 min, and cooled on ice. Four µl of 5x first strand buffer (Invitrogen Japan, Tokyo, Japan), 2 µl of 0.1 M DTT, 0.5 µl of dNTPs (20 mM each dATP, dGTP, and dTTP), 0.48 µl of 100 mM dCTP, 4 µl of [32P]-dCTP, and 200 units of SuperScript II RNaseH- reverse transcriptase (Invitrogen) were mixed and incubated at 42°C for 50 min. The remaining RNA was digested with 2 units of RNaseH (Invitrogen) at 37°C for 20 min. Labeled cDNA was further purified using a QIAquick PCR Purification Kit (QIAGEN).
The membranes were hybridized with labeled target cDNA at 65°C for 16 h with Church phosphate buffer (0.5 M Na2HPO4, 1 mM EDTA, 7% SDS), and washed twice with Church wash buffer (40 mM Na2HPO4, 1 mM EDTA, 1% SDS) for 20 min at 65°C (Church and Gilbert 1984).
Cloning and Northern Hybridization of cDNA of MADS-Box Genes Expressed in Pollen
Complementary DNA of pollen-specific MADS-box genes was amplified by PCR with a poly-T primer (Adaptor primer; Invitrogen), and two nested gene-specific primers located in the MADS domain, using pollen cDNA, and cloned into pAMP1 vector (Invitrogen). These clones were sequenced on an ABI PRISM 377 sequencer using BigDye-terminator chemistry (Applied Biosystems, Foster City, Calif.).
Total RNA extracted from roots collected from juvenile plants grown for 20 days after sowing on an agar plate, juvenile plants with few young rosette leaves without roots grown for 8 days after sowing, mature rosette leaves, inflorescences containing flower buds at stages less than stage 12, flowers at stage 12 and 13, pollens, siliques 0 to 7 days after flowering, and ovules of stage 12 flowers were used. Ten µg of total RNA were electrophoresed on 1% agarose gels containing 1.85% (v/v) formaldehyde and transferred onto Hybond N+ nylon membranes.
The gene-specific probes were partial fragments excluding the MADS domain, amplified by PCR using cDNA as a template. These gene-specific fragments were labeled with [32P]-dCTP using a Random Primer DNA Labeling Kit, version 2.0 (Takara, Tokyo, Japan).
An 802-bp EcoRI fragment of the ROC1 gene (Lippuner et al. 1994) was used as an internal control. The conditions for hybridization and washing were the same as those used for macroarray analyses.
Phylogenetic Analyses
Alignment was performed using ClustalW version 1.8 (Thompson, Higgins, and Gibson 1994). Phylogenetic inferences were made using the Protdist, Neighbor, Seqboot, and Consense programs in the PHYLIP version 3.572c software package (Felsenstein 1995) for the Neighbor-Joining (NJ) analyses. The evolutionary distances were calculated with the Protdist program using the Dayhoff and PAM matrix model (Dayhoff 1978), and a gene tree was constructed by the NJ method (Saitou and Nei 1987) using the program Neighbor. Statistical support for internal branches was estimated by bootstrap analyses with 100 replicates using the programs Seqboot and Consense.
For the maximum likelihood (ML) analyses, the NJdist and ProtML programs in Molphy, version 2.3b3, were used (Adachi and Hasegawa 1996). An NJ tree was obtained with NJdist, based on the ML distance under the JTT model (Jones, Taylor, and Thornton 1992), and used as the starting tree for a local rearrangement search using the program ProtML (Adachi and Hasegawa 1996). The local bootstrap probability of each branch was estimated by the resampling-of-estimated-log-likelihood (RELL) method (Kishino, Miyata, and Hasegawa 1990; Hasegawa and Kishino 1994). Gene names and their accession numbers used for phylogenetic analyses are shown in table 2 of the Supplementary Material online.
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Results |
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Signals for 44 of the 105 putative MADS-box genes were detected in some of the tissues examined, but no signals were detected for the remaining 61 genes (fig. 1). For the latter genes, except MIKCC-type genes, other probes covering the MADS domain were amplified and used for other macroarray analyses to exclude the possibility that the original probe region was not transcribed. The signals of five additional genes were detected with the new probes, and those of the other 56 were not detected (fig. 1). The cDNA sequences of two M-type genes (At5g27130/AGL39 and At4g36590/AGL40) were reported in the EST database. Our macroarray analyses did not detect the expression of either gene even after several repeated experiments.
Characterization of MADS-Box Genes Expressed in Pollen
MADS-box genes expressed in gametophytic tissue are candidates for MADS-box genes with ancestral functions because the ancestors of land plants are inferred to be gametophytic without multicellular sporophytic tissue; sporophytic generation was acquired during land plant evolution (Graham, Cook, and Busse 2000). Female gametophytic tissue, the embryo sac, is embedded in sporophyte tissue and is difficult to isolate, whereas male gametophytes are easy to isolate as pollen grains. The expression of eight MADS-box genes (At2g03060/AGL30, At1g18750, At1g77980, At1g22130, At2g34440/AGL29, At4g11880/AGL14, At3g57390/AGL18, and At5g26950/AGL26) was detected in pollen (fig. 1). Complementary DNA sequences of the previously isolated At4g11880/AGL14 and At3g57390/AGL18 genes were obtained from the DNA database, and the cDNA of other genes, except At5g26950/AGL26, was isolated by 3'-RACE, sequenced, and compared with genomic nucleotide sequences (accession numbers: AB094114, AB094116, AB094111, AB094115, and AB094117). Complementary DNAs of the genes At1g69540 and At1g77950 were also isolated and sequenced (accession numbers: AB094112 and AB094113), because these genes were sisters to At2g03060/AGL30 and At1g77980, respectively, in preliminary phylogenetic analyses (data not shown). We could not isolate At5g26950/AGL26 cDNA, after several attempts, probably because there are genes closely related to At5g26950/AGL26 with more than 90% nucleotide sequence similarity, which prevented PCR amplification during the 3'-RACE analysis.
The At2g03060/AGL30, At1g18750, At1g22130, At1g69540, At1g77950, and At1g77980 genes in the database were predicted to be M-type MADS-box genes, and we did not detect K domains for these genes in the tblastn search using the AG K domain as a query. However, each cDNA, except At1g77950, encodes a weakly conserved K domain based on sequences obtained by 3'-RACE and has an exon/intron structure specific to MIKC*-type MADS-box genes (fig. 1). This is the first record of MIKC*-type genes in seed plants. The stop codon of the At2g03060/AGL30 gene is located in its fifth exon, which encodes the K domain. The At1g77950 gene is composed of a single exon encoding a MADS domain, and it lacks a K domain.
To confirm the expression patterns of these genes, Northern analyses were performed (fig. 3), and the results were concordant with those of the macroarray analyses. Strong signals were detected in pollen for the At2g03060/AGL30, At1g18750, At1g77980, At1g22130, At2g34440/AGL29, and At3g57390/AGL18 genes. Signals of At4g11880/AGL14 mRNA of different sizes were detected in roots and pollen. The mRNA detected in pollen was longer than that detected in root tissue. Weak expression of At3g57390/AGL18 was detected in ovules, which is concordant with previous in situ hybridization studies (Alvarez-Buylla et al. 2000a). Expression of ROC1 (Lippuner et al. 1994), used as an internal control, was detected in all the tissues tested, but the expression was much weaker in pollen than in other tissues. Similar amounts of ribosomal RNA from pollen and other tissues were blotted, indicating that ROC1 mRNA expression is repressed in pollen, and that equal amounts of RNA were blotted.
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The NJ tree of the MADS-box gene family (fig. 4) showed that SRF-type and MEF2-type MADS-box genes form distinct groups. RLM1 and SMP1 of budding yeast MADS-box genes formed a sister group, but they were not sister to either SRF-type or MEF2-type genes. Previously reported moss MIKC*-type MADS-box genes (Henschel et al. 2002) formed a group with the Arabidopsis MIKC*-type genes found in this study (fig. 1). The club moss MIKC*-type gene LAMB1 did not form a group with other MIKC*-type genes. MIKC*-type genes, except LAMB1, were included in MIKCC-type genes, although bootstrap support was low. M-type MADS-box genes were polyphyletic, although some formed monophyletic groups with high bootstrap support.
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Discussion |
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Function of MIKC*-Type Genes
MIKC*-type genes have been reported in a moss and a club moss. Mosses branched from the common ancestor of vascular plants around 400 MYA (Kenrick and Crane 1997). The club moss lineage branched from other extant vascular plants, and then ferns with horsetails (Equisetales) and whisk ferns (Psilotales) diverged from the seed plant lineage (Pryer et al. 2001). Although mosses are an outgroup of Arabidopsis and club mosses, the club moss MIKC*-type gene LAMB1 clustered more basal to Arabidopsis and the moss Physcomitrella MIKC*-type genes (fig. 5) or did not cluster with other MIKC*-type genes in the analysis that used only MADS domain amino acid residues (fig. 4). Although the exon/intron structure of LAMB1 is similar to that of other MIKC*-type genes, LAMB1 likely belongs to another subgroup and is therefore omitted from the following discussion.
All the Arabidopsis MIKC*-type genes except At1g69540 and At1g77950 are specifically expressed in mature pollen, which is gametophytic. Although the expression patterns of moss MIKC*-type genes were not analyzed, these genes were isolated from gametophytic tissues (Henschel et al. 2002), indicating that these moss genes are expressed in gametophyte tissues. An Arabidopsis pollen grain consists of three cells, a vegetative cell and two sperm cells, whereas the Physcomitrella gametophyte is multicellular and forms a shoot-like structure with leaves and stems together with hypha-like protonemata. As angiosperm male gametophytes, such as pollen, are much reduced and the body plans and morphology of the gametophytes of angiosperms and mosses differ, it is difficult to speculate about a common function of MIKC*-type genes in gametophytes of Arabidopsis and Physcomitrella. Analyses of disruptive and over-expressor phenotypes in both Arabidopsis and Physcomitrella are in progress in our laboratories, and the results can be expected to provide further insight into the functions of MIKC*-type genes. At3g57390/AGL18 is a MIKCC-type gene, and it is expressed in both male and female gametophytes (Alvarez-Buylla et al. 2000a), which suggests that this gene has different functions from other MIKC*-type genes.
Classification of MADS-Box Genes Based on Their Phylogeny
Alvarez-Buylla et al. (2000b) inferred the phylogenetic relationships of 45 Arabidopsis MADS-box genes with 9 MEF2-type and 8 SRF-type MADS-box genes, and they found that MADS-box genes are divided into two groups: type I, including all SRF-type genes and 12 M-type genes (At1g65360/AGL23, At1g01530/AGL28, At2g34440/AGL29, At2g03060/AGL30, At2g26320/AGL33, At5g26575/AGL34, At5g26645/AGL35, At5g26625/AGL36, At1g65330/AGL37, At1g65300/AGL38, At5g27130/AGL39, and At4g36590/AGL40), and type II, including all MEF2-type genes and all MIKCC-type genes. They also found some conserved amino acids to support this grouping as synapomorphic characters and stated that an ancestral gene duplication before the divergence of plants and animals gave rise to these two types. In this study, we inferred a phylogenetic tree using 214 MADS-box genes, including all the putative Arabidopsis MADS-box genes (fig. 4); the result necessitates the revision of the type I and II classification. Both MIKCC- and MIKC*-type genes have K domains, indicating the monophyletic relationship of these groups, which is concordant with the phylogenetic tree in figure 4. MEF2-type genes do not form a sister group with MIKCC-type and MIKC*-type genes in figure 4, although the bootstrap support is insufficient and cannot reject the monophyly of MEF2-, MIKCC-, and MIKC*-type genes with statistical confidence. The conserved amino acid residues were also used to support the monophyly of MEF2-type and MIKCC-type genes, and we examined whether these amino acid residues are still conserved in the large data set. The eight amino acid residues R (10th from the start methionine of AP1), Q (18th), V (19th), L (28th), V (37th), D (40th), F (48th), and L (54th) were conserved 8.0 ± 0.2 and 7.2 ± 1.1 in MEF2-type and MIKCC-type genes, respectively. However, these amino acid residues were not well conserved in MIKC*-type genes (5.8 ± 1.6). These results imply that we need to be careful to accept the monophyly of MEF2-, MIKCC-, and MIKC*-type MADS-box genes.
The type I MADS-box genes are probably not monophyletic, according to the phylogenetic tree (fig. 4). At5g26575/AGL34, At5g26625/AGL36, At1g65300/AGL38, and At5g27090 had deletions in the MADS domain and were excluded from the phylogenetic analysis. These genes were closely related to At5g27960, At5g48670, At1g65330/AGL37, and At5g26950/AGL26, respectively, in preliminary phylogenetic analyses. At2g03060/AGL30 has been classified as a type I gene, but our comparison of its cDNA to genomic sequences showed that At2g03060/AGL30 is a MIKC*-type gene, even though there is a stop codon in the 5th exon, suggesting that AGL30 protein lacks the K and C domains, if translated. We could not assign I, K, and C domains for other type I MADS-box genes. These genes were polyphyletic and were not sister to SRF-type genes (fig. 4), indicating that the grouping of type I MADS-box genes is inappropriate.
Because the original grouping of type II MADS-box genes did not include MIKC*-type genes, and because the monophyly of type I genes is not supported, it is better to use the original classification based on the domain structure: SRF-, MEF2-, MIKCC-, MIKC*-, and M-types. Each of the first four types likely has a single origin, but the last does not. The phylogenetic relationships among the first four groups were not resolved with statistical confidence, and future studies are necessary. RLM1 and SMP1 of yeast MADS-box genes did not cluster with either SRF- or MEF2-type genes, suggesting that these genes form a new group of MADS-box genes.
Origin and Evolution of M-Type MADS-Box Genes
The most unexpected result of this study was that most M-type MADS-box genes were not expressed in any tissues examined. We used tissues that covered most developmental stages; most M-type genes probably do not function in regular development in Arabidopsis. Furthermore, the branch lengths of most M-type MADS-box genes are relatively longer than those of other types of genes, suggesting that the functional constraints on most M-type genes are not as strong as in other genes, and that M-type genes are pseudogenes. We should be careful in regarding all M-type genes as pseudogenes and non-functional, because some M-type genes are expressed (fig. 1), and the amino acids are conserved in some clades (e.g., the clade including At4g36590/AGL40 in fig. 4).
The conspicuous shared character of M-type genes is that these genes are intron-less, or have few introns, based on the computer prediction in the MIPS site (fig. 1), which suggests that M-type genes are functional retrogenes or processed retropseudogenes (Weiner, Deininger, and Efstratiadis 1986). Retrogenes are reverse-transcribed from mRNA and usually have poly-A sequences at their 3' region. Such poly-A sequences were not found within 1 kb of the predicted stop codons, except in At5g49490 (data not shown), indicating that most M-type genes are probably not retropseudogenes or retrogenes. There are 25 adenine nucleotides at the 183rd site from the stop codon of At5g49490, suggesting that it is a retrogene. Another possible origin of M-type MADS-box genes is that they are pseudogenes that originated from MIKCC-type or MIKC*-type genes. The K domain is characterized by regular spacing of conserved hydrophobic residues and is inferred to form amphipathic helices (Shore and Sharrocks 1995). The MADS domain is composed of ca. 60 conserved amino acid residues, and might still be distinguished after substantial mutations accumulate. The MADS domain is distinguishable even after a stop codon is formed in the MADS domain as in At3g32313, At3g32316, T8N9.10, At3g32371, and At3g31902. By contrast, approximately 10 amino acid residues characterized only by their hydrophobicity, which are spaced throughout the K domain, are weakly conserved, and it would be difficult to recognize a K domain with the same number of accumulated mutations. In other words, once MIKCC-type or MIKC*-type genes became pseudogenes, the K domain would be lost faster than the MADS-domain. At1g77950 might be an example of this process; it was transcribed as an M-type gene, but the exon/intron structure of MIKC*-type genes in region I remained in the genomic sequence. No K domain was found, even with a careful search of the amino acid sequences in the region corresponding to the K domain of At1g77950 (data not shown). At1g77950 probably originated in a duplication of its sister gene, At1g77980, which is expressed and is likely functional. If M-type MADS-box genes are pseudogenes that originated by duplications of functional MIKCC-type or MIKC*-type genes, it is curious that most M-type MADS-box genes do not have corresponding sister genes, which should form sister groups in the phylogenetic tree (fig. 4). One possible explanation is that after substantial mutations randomly accumulate in a pseudogene, following gene duplication, the pseudogene does not form a sister group with its sister gene, and likely clusters with other pseudogenes as a result of long-branch-attraction (Felsenstein 1978). Note that some M-type MADS-box genes are duplicated repeatedly, and the numbers increase after the original pseudogenes fully diverge from their ancestor (e.g., the clade including At5g49490). Once MIKCC-type or MIKC*-type genes become pseudogenes, how long can we recognize these pseudogenes as MADS-box genes? There are approximately two synonymous nucleotide substitutions per site between eudicots and monocots (Wolfe, Sharp, and Li 1989). When nucleotides are substituted under a Poisson distribution (Sokal and Rohlf 1995, p. 84), approximately 86% of their nucleotides should be substituted more than once after the divergence of eudicots and monocots, approximately 200 MYA, implying that most amino acids in the MADS domain were probably substituted more than once. The BLAST search that we used in this study obtained MADS-box genes containing more than 20% amino acid sequences identical to the At4g18960/AG gene. A rough calculation indicates that a pseudogene that originated at the time of eudicot/monocot divergence would not be included in this study. If the M-type genes detected in this study are pseudogenes, they originated after the divergence of eudicots and monocots.
The expression patterns of previously characterized MADS-box genes (reviewed in Johansen et al. 2002) are concordant with the results shown in figure 1. This indicates that our method is highly reliable. However, the sensitivity of our method of detecting the expression of mRNA is not as high as that of reverse-transcription-PCR (RT-PCR), and we cannot deny the possibility that M-type genes whose expression was not detected in this study may be expressed at very low levels and be functional. We performed RT-PCR for all genes whose expression was not detected in figure 1, and detected the expression of the following genes (data not shown): At1g17310, At1g28460, At1g31140, At1g31630, At1g46408, At1g47760, At1g48150, At1g54760, At1g59810, At1g60880, At1g60920, At1g65300, At1g65330, At1g65360, At2g24840, At2g26320, At2g26880, At2g28700, At2g40210, At3g05860, At3g66656, AtMADS3, AtMADS4, At4g36590, AtMADS5, At5g06500, At5g26625, At5g26645, At5g27130, At5g27580, At5g27960, At5g38620, At5g39750, At5g40120, At5g41200, At5g48670, At5g49420, At5g49490, At5g55690, At5g58890, At5g60440, and At5g65330. Further studies using loss-of-function mutants of these genes are necessary to analyze whether these genes are functional. It should also be noted that several M-type MADS-box genes might be induced by environmental factors.
Evolution of the Expression Patterns of MIKCC-Type MADS-Box Genes
The expression patterns of all the MADS-box genes in the Arabidopsis genome were analyzed using the macroarray technique (fig. 1), and those of eight genes were confirmed by Northern analysis (fig. 3). The expression patterns of most MIKCC-type genes are concordant with those previously reported (Alvarez-Buylla et al. 2000a), except AGL19. Weak expression of AGL19 was detected in rosette leaves, but not in roots, although Alvarez-Buylla et al. (2000a) detected AGL19 expression in roots in Northern and in situ RNA analyses. This difference is probably caused by the probe used in the previous study, which was a whole cDNA, including the MADS domain. The nucleotide sequence of the AGL19 MADS-box is 90% identical to that of At4g11880/AGL14, which is expressed in roots, suggesting cross-hybridization of the probe to At4g11880/AGL14 mRNA in roots.
Our phylogenetic analyses confirmed a previous result, which was that MIKCC- type MADS-box genes are divided into several monophyletic groups, and that members of each group are similarly expressed (reviewed in Theissen et al. 2000). The evolution of the expression patterns should be discussed according to the phylogenetic relationships of genes with reliable statistical confidence. In the case of a MADS-box gene family, inter-subfamily relationships are not inferred with statistical support, whereas the monophyletic relationship of each subfamily is usually supported with high bootstrap values (Theissen et al. 2000; Johansen et al. 2002). Some authors have stated that MADS-box genes expressed in vegetative organs predate those in reproductive organs; in other words, reproductive MADS-box genes originated from duplicated vegetative MADS-box genes (Purugganan 1997; Alvarez-Buylla et al. 2000a). However, the phylogenetic relationships used in those studies have not been reproducible in larger-scale analyses (Theissen et al. 2000; Johansen et al. 2002), including this study, and we must state that the evolution of expression patterns in MIKCC-type genes is still ambiguous. Further analyses including a larger number of genes from other taxa may give more robust phylogenetic trees to infer the evolution of expression patterns in the MADS-box gene family.
Evolution of Gametophytic and Sporophytic MADS-Box Genes
Based on fossil data, land plants emerged approximately 480 MYA (Kenrick and Crane 1997), and both morphological and molecular data support the sister relationship between extant land plants and the freshwater green algae, charophytes (Graham, Cook, and Busse 2000; Graham and Wilcox 2000, p. 497; Karol et al. 2001). Land plants are composed of four monophyletic groups: vascular plants, mosses, liverworts, and hornworts. The phylogenetic relationships of these groups are still controversial, because of discrepancies among the phylogenetic relationships inferred from morphological and molecular data and among the different molecular data sets used (Nishiyama and Kato 1999; Qiu and Palmer 1999; Nickrent et al. 2000). However, there is consensus that bryophytes, which include the last three groups, are more basal than vascular plants in the land plant phylogenetic tree. Bryophytes are characterized by their dominant gametophytic plant body and their sporophytes epiphytic to gametophytes. The sporophytes of bryophytes do not differentiate shoots and roots, unlike vascular plants, but form only a sporangium with tissue connecting them to the gametophytes. In charophytes, the first cell division of the zygote is meiosis, and their plant body is gametophytic. A sister group of the charophyte-land plant clade is the zygnematalean green algae, which have a life cycle similar to that of charophytes (Graham and Wilcox 2000, p. 503). These features suggest that the common ancestor of charophytes and land plants had a life cycle similar to that of charophytes, and that the common ancestor of bryophytes and vascular plants was gametophyte-dominant. According to this evolutionary scenario of land plants, all the genes used in the development of the diploid plant body should have originated from those used in gametophytes. Expression analyses of all the Arabidopsis MADS-box genes showed that most MIKCC-type genes, except At3g57390/AGL18, were not expressed, or were very weakly expressed, in the male gametophyte, whereas all MIKC*-type genes, except At1g69540, were specifically expressed in the male gametophyte. This suggests that At3g57390/AGL18 and the MIKC*-type genes retain their original function in gametophytes from the common ancestor of land plants and charophytes. The characterization of these genes will give further insight into the original function and subsequent evolution of MADS-box genes. DEFH125 of Antirrhinum and ZmMADS2 of Zea MADS-box genes are expressed in pollen (Zachgo, Saedler, and Schwarz-Sommer 1997; Heuer, Lörz, and Dresselhaus 2000). Phylogenetic analyses including these taxa showed that both genes formed a clade with At2g22630/AGL17, At4g37940/AGL21, At3g57230/AGL16, and At2g14210/ANR1 (figs. 4 and 5), which are expressed in sporophytes (fig. 1). This suggests that these genes were secondarily recruited for expression in the gametophyte from sporophyte genes. Only MIKCC-type genes are reported from ferns, and they are expressed in both the gametophyte and sporophyte (Münster et al. 1997; Hasebe et al. 1998), suggesting that MIKCC-type genes expanded their expression from the gametophyte to the sporophyte before the common ancestor of ferns and angiosperms, and that the MIKCC-type genes, except At3g57390/AGL18, lost their expression in the gametophyte. Both MIKCC- and MIKC*-type genes are found in the moss Physcomitrella gametophyte, and the common ancestor of mosses and angiosperms should have expressed both types in gametophytes. These results indicate that dynamic changes in gene expression between gametophyte and sporophyte occurred during land plant history and may be correlated with the evolution of body plan.
This evolutionary model lacks information on gene expression in the female gametophyte in angiosperms. The only MADS-box gene reported to be expressed in the female gametophyte, the embryo sac, is At3g57390/AGL18 (Alvarez-Buylla et al. 2000a), and more detailed analyses of Arabidopsis MADS-box genes in this tissue will give further insights into this model.
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