© 2007 The Author(s).
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RESEARCH PAPER |
The conserved cysteine-rich domain of a tesmin/TSO1-like protein binds zinc in vitro and TSO1 is required for both male and female fertility in Arabidopsis thaliana
1Laboratory of Gene Expression, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark
2BiRC–Bioinformatics Research Center, University of Aarhus, Ny Munkegade Building 540, DK-8000 Aarhus C, Denmark
3Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
To whom correspondence should be addressed. E-mail: stig.andersen{at}tuebingen.mpg.de
Received 19 March 2007; Revised 30 July 2007 Accepted 16 August 2007
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Abstract |
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Development of reproductive tissue and control of cell division are common challenges to all sexually reproducing eukaryotes. The Arabidopsis thaliana TSO1 gene is involved in both these processes. Mild tso1 mutant alleles influence only ovule development, whereas strong alleles have an effect on all floral tissues and cause cell division defects. The tso1 mutants described so far carry point mutations in a conserved cysteine-rich domain, the CRC domain, but the reason for the range of phenotypes observed is poorly understood. In the present study, the tesmin/TSO1-like CXC (TCX) proteins are characterized at the biochemical, genomic, transcriptomic, and functional level to address this question. It is shown that the CRC domain binds zinc, offering an explanation for the severity of tso1 alleles where cysteine residues are affected. In addition, the phylogenetic and expression analysis of the TCX genes suggested an overlap in function between AtTSO1 and the related gene AtTCX2. Their expression ratios indicated that pollen, in addition to ovules, would be sensitive to loss of TSO1 function. This was confirmed by analysis of novel tso1 T-DNA insertion alleles where the development of both pollen and ovules was affected.
Key words: Meristem, ovule, pollen, retinoblastoma, SynMuv
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Introduction |
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Most animals and flowering plants are capable of sexual reproduction. During this process haploid germ cells have to be produced, and the development of specialized organs, which facilitate germ cell production, is a common challenge to plants and animals. Female germ cells are produced in the ovary. In flowering plants, the ovary is embedded in the carpel and contains ovules, which develop into seeds after fertilization. Male germ cells are known as sperm cells, and are produced in the testes of animals and anthers of plants, respectively.
The human TESMIN gene was originally identified by its specific expression in testes, but subsequently it was also detected at specific stages of ovary development (Sugihara et al., 1999; Sutou et al., 2003; Olesen et al., 2004). Concomitantly, mutant alleles of the Arabidopsis thaliana TSO1 gene causing defects in flower and ovule development were identified (Hauser et al., 2000; Song et al., 2000). ‘Tso’ means ‘ugly’ in Chinese and refers to the appearance of tso1 mutant flowers. Both Hstesmin and AtTSO1 localize to the nucleus and contain two cysteine-rich CXC motifs (pfam03638) with the consensus sequence CXCX4CX3YCXCX6CX3CXCX2C separated by a region of variable length containing the short conserved sequence RNPXAFXPK (Fig. 1A) (Cvitanich et al., 2000; Song et al., 2000; Sutou et al., 2003). This domain has previously been named the CRC domain, the TCR motif, and the CHC domain (Cvitanich et al., 2000; Song et al., 2000; Sutou et al., 2003). Here, the term CRC domain will be used, and proteins containing the CRC domain will be called TCX proteins. As previously noted (Cvitanich et al., 2000; Hauser et al., 2000; Song et al., 2000), the cysteine-rich domains most similar to the CRC domain are found in the Drosophila melanogaster Enhancer of zeste [E(z)] protein and its homologues. In A. thaliana these include CURLY LEAF (Goodrich et al., 1997; Kim et al., 1998) and MEDEA (Grossniklaus et al., 1998; Kiyosue et al., 1999). These proteins belong to the polycomb group of proteins, which are involved in maintaining stable repression of gene expression through cell divisions and have functions in the regulation of cell proliferation (Reyes and Grossniklaus, 2003). Despite the similarity between TCX and E(z) type proteins, a clear distinction can be made since E(z) proteins lack the RNPXAFXPK motif (Fig. 1A, B).
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Recently, evidence from D. melanogaster has suggested that functional similarities exist between TCX and polycomb group proteins. The DmTCX1 (dMip120) protein is part of a protein complex that binds to specific replication origins located at chorion gene clusters (Beall et al., 2002). DmTCX1 and other members of the replication origin-binding complex also interact with retinoblastoma (RB) and E2F proteins in complexes named dREAM (Drosophila RB, E2F, and Myb) and Myb–MuvB, respectively (Korenjak et al., 2004; Lewis et al., 2004). Like polycomb protein complexes, dREAM complexes associate with chromatin that is not actively transcribed (Korenjak et al., 2004). While dREAM and polycomb complexes both bind to transcriptionally inactive regions, their binding sites do not overlap (Korenjak et al., 2004). Different types of dREAM complexes regulate the expression of specific subsets of genes including a group involved in cell division and genes with developmental- or sex-specific expression patterns (Korenjak et al., 2004). TCX proteins have also been described in Caenorhabditis elegans where homologues of dREAM complex members belong to the synthetic multivulva (SynMuv) class B genes. When mutant alleles of class B SynMuv genes are combined with mutations in class A or C SynMuv genes, the affected worms develop an abnormally high number of vulvae (Ceol and Horvitz, 2001, 2004), and it has been shown that the C. elegans CeTCX1 (JC8.6) gene belongs to the SynMuv class B genes (Owen et al., 2003; Korenjak et al., 2004).
Thus, there is evidence that animal TCX proteins are associated with development of both male and female reproductive tissues. In plants, the mild A. thaliana tso1-3 and tso1-4 mutations affect only ovule development. This phenotype indicates that AtTSO1 function could be restricted to female reproductive tissue (Hauser et al., 2000). However, the strong tso1-1 and tso1-2 alleles affect all floral tissues, both sterile and reproductive, as well as inflorescence meristem organization and cell division. Why mutations within the same gene cause such diverse effects is poorly understood. In the present study, this question is addressed by characterizing the TCX protein family at the biochemical, genomic, transcriptomic, and functional level. It is suggested that the range of phenotypes observed can be attributed to zinc binding by the CRC domain and an overlap in function between AtTSO1 and the closely related AtTCX2 protein. Furthermore, through analysis of novel tso1 mutant alleles, it is shown that AtTSO1, like many of its animal TCX gene counterparts, is associated with the development of both male and female reproductive tissue.
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Materials and methods |
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Sequence retrieval and alignment, and phylogenetic tree construction
TCX protein sequences were retrieved using BLAST (Altschul et al., 1990) to search the GenBank translated genome databases, organism by organism, with the CRC domain protein sequence of AtTSO1 as subject. In addition, all proteins discovered within one organism were used as BLAST subjects in that same organism. Protein coding regions in rice were predicted using Twinscan and Genebuilder programs (Milanesi et al., 1999; Korf et al., 2001). All alignments were performed with the T-Coffee alignment program (Notredame et al., 2000) using the web interface available at CNRS, Marseille, France (http://igs-server.cnrs-mrs.fr) with standard options. The phylogenetic analysis of CRC domains from TCX proteins was conducted using MEGA version 2.1 (Kumar et al., 2001). The phylogenetic tree was constructed using the minimal evolution method with Poisson correction for amino acid distance, and handling gap/missing data by pair-wise deletion. Confidence values were obtained by 1000-fold bootstrap tests.
Recombinant protein purification, and metal and amino acid analysis
The construct for expression of the glutathione S-transferase (GST)–CRC fusion protein was described previously (Cvitanich et al., 2000) and the pGEX-5x-3 was used for GST expression. Escherichia coli BL21 cultures were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 °C for 2–5 h. Cells were harvested and resuspended in 0.8 M NaCl, 50 mM HEPES pH 7.5, 5 mM benzamidine-HCl, 10% (v/v) glycerol, 5 mM β-mercaptoethanol, and 0.5 M urea. Cells were lysed by sonication and ultracentrifuged at 100 000 g for 3 h at 4 °C. The supernatant was incubated with glutathione–Sepharose 4B beads (Amersham Biosciences, Uppsala, Sweden) for 30 min at room temperature. The beads were washed with phosphate-buffered saline (PBS) containing 0.8 M NaCl, 0.1% (v/v) Triton X-100, 0.5 M urea, and 5 mM β-mercaptoethanol, and fusion protein was eluted with a buffer containing 10 mM reduced glutathione, 50 mM TRIS-HCl pH 8.0, 2 M urea, 10 mM benzamidine-HCl, and 5 mM β-mercaptoethanol. Approximately 1 mg of protein was sent to West Coast Analytical Service (Santa Fe Springs, CA, USA, http://www.wcas.com) for inductively coupled plasma mass spectrometry analysis. Protein for amino acid analysis was hydrolysed for 16 h at 110 °C in 6 M HCl, 0.05% (v/v) phenol. For zincon (zincon sodium salt hydrate, FLUKA 33826) measurements, protein was brought into 10 mM NH4HCO3 using a Sephadex G-25 fine column, and degraded with proteinase K overnight before incubating with 125 µM zincon dye in 10 mM NH4HCO3. Colour formation was measured at 620 nm.
Plant materials and RT-PCR expression analysis
For RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis of specific tissues, A. thaliana ecotype Landsberg erecta was used. Microspores, pollen, leaves, and stems were harvested from plants grown in controlled-environment cabinets at 21 °C under illumination of 100 µmol m–2 s–1 with a 16 h photoperiod. Roots were taken from plants grown in the dark at 22 °C in 0.5x MS medium (Sigma, Steinheim, Germany) supplemented with 1% (w/v) sucrose under constant shaking for 6 weeks. Mature pollen and isolated spores from three developmental stages were harvested from two independently grown plant populations as described (Honys and Twell, 2003, 2004). Total RNA was isolated with the RNeasy plant RNA isolation kit (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized from 1 µg of total RNA using the Improm II reverse transcription system (Promega, Madison, WI, USA), and 1 µl of 50x diluted cDNA was used as a template in RT-PCR analysis. Gene-specific primers spanning introns were designed for each AtTCX gene and AtKAPP; their sequences are listed in Supplementary Table S2 available at JXB online. PCR conditions were as follows: 95 °C for 30 s followed by 40 cycles of 94 °C (15 s), 55 °C (30 s), and 72 °C (30 s), except for AtKAPP where an annealing temperature of 50 °C was used.
Insertion mutant plant lines were ordered through the National Arabidopsis Stock Center (NASC): Attso1-5 (SALK_102956, CC884124, At3g22780), Attso1-6 (SALK_074231, BH901280, At3g22780), and Attcx3-1 (GT_3_37840, BX537478, At3g22760) (NASC line name, GenBank accession number, and TAIR locus identifier, respectively) (Sundaresan et al., 1995; Alonso et al., 2003). Insertion mutant genotyping was done by standard PCR using sets of primers amplifying either the wild-type or mutant allele (Supplementary Table S3 at JXB online). Plants were grown in controlled-environment cabinets at 21 °C under illumination of 100 µmol m–2 s–1 with a 16 h photoperiod on Fiboklinker (Optiroc, Randers, Denmark) and watered with Hornum fertilizer [0.5% (v/v)] or in continuous light on soil. For RNA extraction from insertion mutant lines, the RNeasy plant RNA isolation kit (Qiagen, Valencia, CA, USA) was used to extract RNA from inflorescences. cDNA was synthesized using reverse transcriptase (Fermentas). RT-PCR was carried out with a 5x dilution of the cDNA product as template using a touchdown PCR program: 10 cycles of 95 °C for 30 s, 65 °C for 30 s (–1 °C per cycle), 72 °C for 40 s, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 40 s, and 72 °C for 4 min. The housekeeping gene ADENINE PHOSPHORIBOSYLTRANSFERASE (AtAPT) (Moffatt et al., 1994) was used as control. The position of the primers used for RT-PCR analysis is indicated in Fig. 4A, and primer sequences can be found in Supplementary Table S2 at JXB online.
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Analysis of Affymetrix microarray data
Affymetrix expression data for AtTSO1, AtTCX2, AtTCX3, AtTCX5, AtTCX6, and AtTCX8 were downloaded from the NASC (Craigon et al., 2004) Affymetrix database using the Bulk Gene Download tool. Signal values corresponding to transcripts called absent were set to zero. Experiments on plant species other than A. thaliana were removed from the data set. The slide named ‘Casson_1-9_heart-stage-cotyledon_Rep3_ATH1’ presented an unrealistically high expression value for AtTXC2 when compared with the other two replicates and was omitted from the analysis. The resulting data set was used for all subsequent calculations (Table 2, and Supplementary Table S1 at JXB online). The average expression signal across all experiments was calculated for each gene, and TCX expression signals were related to this average. Experimental slides in which at least one of the TCX genes was expressed at levels >2-fold the average value were selected for further analysis. Slides that were not part of a group of at least two replicates were discarded. A complete list of selected experimental slides can be found in Supplementary Table S4 at JXB online. The remaining slides were grouped in sets of replicates, and average values were calculated for each group. Groups displaying data from A. thaliana ecotypes Columbia and Landsberg erecta were selected. These were then sorted by tissue and are presented in Table 2.
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Histochemical staining
Nuclei in freshly collected pollen were stained with propidium iodide [0.1% (v/v), Fluka] in PEM buffer (0.05 M PIPES, 1 mM EGTA, 0.5 mM MgCl2, pH 6.9) with 0.01% (v/v) Triton X-100, for 30 min at room temperature. Pollen grains were observed under a Zeiss 510 Meta confocal microscope. β-Glucuronidase (GUS)-expressing tissue was stained as follows: flowers were incubated in the staining solution [0.05% (w/v) X-Gluc; 0.05 M NaPO4 pH 7.0; 0.02 M EDTA; 20% (v/v) methanol] overnight at 37 °C. Subsequently the tissue was cleared overnight in 70% ethanol and viewed under a stereomicroscope. Plastic embedding, sectioning, and staining of floral tissue were carried out as previously described (Andersen et al., 2003).
Accession numbers
The A. thaliana genes mentioned in this study have the following Arabidopsis Genome Initiative (AGI) identifiers: APT (ADENINE PHOSPHORIBOSYLTRANSFERASE, At1g27450), CLF (CURLY LEAF, At2g23380), KAPP (KINASE-ASSOCIATED PROTEIN PHOSPHATASE, At5g19280), MEA (MEDEA, At1g02580), and RBR (RETINOBLASTOMA-RELATED, At4g16130). DmE(z) protein has the GenBank accession number P42124. Accession numbers for the remainder of the genes can be found in Table 1.
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The CRC cysteine-rich domain binds zinc in vitro
All tso1 mutants described so far (Hauser et al., 2000; Song et al., 2000) harbour point mutations in the cysteine-rich CRC domain, which defines the TCX protein family. The sensitivity to mutations and the strict cross-species conservation suggest that the CRC domain is critical for TCX protein function. Although the cysteine pattern of the CRC domain does not correspond to any known metal-binding motif, the conservation of cysteine residue positions hints that the domain could co-ordinate metal ions.
To investigate this possibility, a purified GST–CRC fusion protein was subjected to inductively coupled plasma mass spectrometry. Zinc was the only metal for which a substantial difference in metal content was observed between the buffer blank and the protein sample (Fig. 1C). The data indicated that each GST–CRC molecule co-ordinates at least four zinc ions, since a difference in zinc content of 4.2 µg g–1 was observed, corresponding to a zinc concentration of 64 µM. By Bradford and amino acid analysis, the protein concentration was determined to be 19 µM and the estimated protein purity was 85% based on the SDS–gel electrophoresis. However, it cannot be ruled out that more zinc can be bound by GST–CRC under certain conditions and, therefore, four should be considered the minimal number of zinc ions co-ordinated.
To rule out that GST binds zinc under the present experimental conditions, the zinc-binding dye zincon was used to probe the zinc content of GST and GST–CRC proteins expressed, purified, and degraded in parallel. Despite digesting excess GST, zinc could be detected exclusively in the GST–CRC sample (Fig. 1D). Zinc was only detected after digestion, indicating that it is tightly associated with the GST–CRC protein.
AtTSO1 belongs to the plant-specific type 1 TCX proteins
It has been noted that two TCX proteins similar to TSO1 exist in A. thaliana (Hauser et al., 2000; Song et al., 2000). These and other TCX family members may be the key to understanding the variation in tso1 mutant phenotypes. In addition, identification of TCX proteins from other organisms could provide further insights into TCX protein function. To recover the full complement of TCX proteins in A. thaliana and to get a representative overview of similar proteins in other organisms, completely sequenced genomes were searched for TCX family members. The organisms were chosen so that plants, mammals, fungi, nematodes, amoebae, and prokaryotes were all represented. A total of 26 TCX loci from A. thaliana, Oryza sativa, Homo sapiens, Mus musculus, D. melanogaster, C. elegans, and Dictyostelium discoideum were identified using BLAST (Altschul et al., 1990) searches in GenBank (Table 1). No TCX proteins were found in prokaryotes and fungi. Evidence of expression in the form of expressed sequence tag (EST) or mRNA sequences was available for 20 of the 26 loci. Eleven TCX loci were found in O. sativa, making it the organism with the largest number of TCX family members. Arabidopsis thaliana had eight TCX loci, while only one or two TCX loci per genome were found in animals.
The CRC domains of all identified TCX proteins were aligned (Fig. 1E) and the alignment revealed a high level of conservation across the eukaryotic kingdom. Not only the cysteines, but also a number of intervening residues, were completely invariant across all investigated organisms. To determine the evolutionary relationships between TCX proteins, the alignment of CRC domains (Fig. 1E) was used as the basis for a phylogenetic analysis. The resulting tree (Fig. 2A) indicated that TCX proteins could be divided into distinct phylogenetic groups. To confirm the validity of the tree, full-length protein alignments were made for the putative groups. Type-specific conserved sequences were identified within three of these (Fig. 2B, C). No additional domains with known functions were present. Based on the phylogenetic tree and the type-specific conserved motifs, TCX proteins could be divided into three robust subgroups, types 1, 2, and 3 (Fig. 2). Type 1 and type 2 TCX proteins are present in both A. thaliana and O. sativa. The animal TCX proteins make up the third group, type 3. Several TCX sequences from O. sativa do not belong to any of the subgroups (types 1, 2, and 3). The DmE(z) protein was used as an outgroup, but the evolutionary relationship of types 1, 2, and 3 TCX proteins is not clear from the tree, since the confidence levels of the nodes are low at higher hierarchical levels. However, the closest relatives of the only TCX protein found in the primitive eukaryote D. discoideum are the type 2 TCX proteins. This suggests that type 1 and type 2 TCX proteins arose through a plant-specific duplication and that type 2 TCX proteins have diverged least from the ancestor. AtTSO1 belongs to the type 1 TCX proteins and has two close type 1 homologues, AtTCX2 and AtTCX3, as well as the more distantly related type 1 protein AtTCX4.
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The expression patterns of AtTSO1 and AtTCX2 are similar
To compare the expression pattern of AtTSO1 with the expression profiles of other A. thaliana TCX genes, transcription analyses were performed. First, an overview of TCX gene expression in different tissues was obtained using RT-PCR (Fig. 3). No transcripts from the AtTCX4, AtTCX7, and AtTCX8 genes were detected in any of the tissues examined. Control PCRs using genomic DNA showed that the primers chosen did amplify the target genes (data not shown). While the type 2 genes AtTCX5 and AtTCX6 showed relatively similar expression levels across the tissues investigated, large variation was seen for the type 1 genes AtTSO1, AtTCX2, and AtTCX3. The most obvious difference was the high expression level of AtTCX3 in all stages of pollen development compared with that of AtTSO1 and AtTCX2 (Fig. 3). It is also noticeable that the AtTSO1 transcript was found in uninucleate microspores and bicellular pollen but not in tricellular and mature pollen (Fig. 3).
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To elaborate on the RT-PCR expression analysis, the Affymetrix microarray data available at NASC (Craigon et al., 2004) were analysed. The data downloaded included expression information for AtTSO1, AtTCX2, AtTCX3, AtTCX5, and AtTCX6 in 1826 experiments conducted with the Affymetrix GeneChip ATH1 (Craigon et al., 2004; Redman et al., 2004; Schmid et al., 2005). Microarray slides were selected based on the expression signal of TCX genes (see Materials and methods for details), and analysis of these experiments revealed that TCX genes are highly expressed in the shoot apex, carpels, pollen, and seeds (Table 2).
In the shoot apex, AtTCX2 was highly expressed at all developmental stages. AtTSO1 expression increased as the vegetative shoot meristem changed into the inflorescence meristem (bolted), whereas AtTCX3 expression displayed the opposite trend—decreasing from a high expression value before bolting to a lower value after the transition. This trend was observed in three independent experimental series (Table 2, 1–13).
In flowers, AtTCX3 expression dominated in the male part of the flower due to its very high expression in pollen (Table 2, 16–17 and 20–24). Confirming the RT-PCR experiments (Fig. 3), AtTSO1 transcripts were detected in uninucleate and bicellular pollen but not in tricellular and mature pollen (Table 2, 20–24).
The data presented in Table 2 showed that AtTCX2 shares a high degree of similarity in gene expression pattern with AtTSO1. To investigate this quantitatively, Pearson correlation coefficients were calculated based on the complete data set. AtTSO1 and AtTCX2 had the highest correlation coefficient (0.66) of all TCX gene pair comparisons, confirming that AtTCX2 has the most similar expression pattern to AtTSO1 among the TCX genes. Pearson correlation coefficients for all comparisons are given in Supplementary Table S1 at JXB online.
AtTSO1 is necessary for correct development of pollen, carpels, and ovules
The tso1 mutants characterized so far all harbour point mutations (Hauser et al., 2000; Song et al., 2000). In order to compare the phenotypes of these mutants with those of mutants carrying insertions in type 1 TCX genes, the T-DNA Express (Alonso et al., 2003) database was searched. Three mutant lines, tso1-5, tso1-6, and tcx3-1, were identified, in which the insertions affected TCX gene expression. Insert positions were confirmed by sequencing, and their position relative to exons and conserved domains are illustrated in Fig. 4A. No reduction in transcript levels 5' of the insertion was seen for tso1-5 and tso1-6 (Fig. 4B). In contrast, a significant reduction in transcript level 3' of the insertion site was observed for all three mutant lines (Fig. 4B).
Despite AtTCX3 normally being highly expressed in pollen, no phenotypic changes were apparent for tcx3-1 mutants in this tissue. The tcx3-1 mutant is a gene trap line, and GUS activity was detected in pollen (Fig. 5T). However, the activity level varied greatly between plants, with no apparent correlation to pollen developmental stage.
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Both tso1-5 and tso1-6 homozygous mutants had shorter siliques than wild-type plants. Heterozygous plants were indistinguishable from the wild type. tso1-6 plants were sterile, whereas tso1-5 plants produced very few seeds. Sepals and petals looked largely normal, though the sepals had serrated edges in the tso1-6 mutant (Fig. 5C). The most obvious phenotypic deviations were found in the carpels. In the tso1-5 mutant, stigmatic papillae grew longer than in the wild type (Fig. 5B). In tso1-6 mutants, the carpels usually failed to fuse properly, causing a serrated structure with excessive growth of stigmatic papillae (Fig. 5C). The lack of proper fusion was also apparent in the developing siliques of both lines (Fig. 5F, G). For tso1-6, it was often so severe that the ovules were visible. In both tso1-5 and tso1-6 mutants the shape of the outer integument cells was highly variable, the shape of the ovules was aberrant, and the embryo sac was often absent (Fig. 5L). These defects in ovule development are similar to those previously described for the tso1-3 mutant (Hauser et al., 1998).
The overall morphology of tso1-5 and tso1-6 anthers was normal. However, very little pollen was deposited on the stigma and pistil. A closer investigation revealed that anthers from both mutants contained two types of pollen grains. Some were large, yellow, and rounded, whereas the rest were small, brown, and collapsed (Fig. 5N–P). In the tso1-6 mutant, most pollen grains were collapsed, while fewer pollen grains displayed this phenotype in the tso1-5 mutant. In the expression analysis, AtTSO1 transcript was only detected at the stages of pollen development where cell divisions are taking place. To determine if pollen cell divisions were progressing normally in the absence of AtTSO1, the number of nuclei in mutant pollen grains was analysed. Propidium iodide staining showed that the large tso1-5 and tso1-6 pollen grains contained two strongly staining sperm cell nuclei and a single diffuse vegetative cell nucleus as in wild-type pollen (Fig. 5Q–S). The staining intensities and sizes of wild-type, tso1-5, and tso1-6 pollen nuclei were quantified by confocal microscopy, but no significant differences were observed between the different genotypes. No aberrant pollen phenotypes were observed in tso1-5 and tso1-6 heterozygous plants, and the segregation ratio of the tso1 alleles was normal. This suggests that the effect of TSO1 on pollen development is sporophytic.
Heterozygous tso1-5 and tso1-6 plants were crossed to confirm that the two mutations were allelic. Out of the resulting 102 plants, 25 were tso1-5/tso1-6 heteroallelic and they all displayed the mutant phenotype (Fig. 5D, H, P). Plants with all other genotypes were indistinguishable from the wild type.
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The TCX protein family
TCX proteins have previously been described in several eukaryotic organisms (Sugihara et al., 1999; Cvitanich et al., 2000; Hauser et al., 2000; Song et al., 2000; Beall et al., 2002; Owen et al., 2003; Korenjak et al., 2004; Lewis et al., 2004). The present phylogenetic analysis has shown that TCX proteins are absent in prokaryotes but present in all eukaryotes investigated, with the notable exception of fungi. The CRC domains of TCX proteins show high conservation across species from amoebae through plants to mammals. The zinc co-ordination by this domain most probably restricts sequence variation. In contrast, only little sequence similarity was found outside the CRC domain, which is the only conserved domain present in all TCX proteins.
Large differences in the number of TCX loci between organisms were found. In particular, plants contain a higher number of TCX genes than animals. Eight and 11 TCX proteins were found in the A. thaliana and O. sativa genomes, respectively, whereas animals contain one or two TCX loci. The type 1 and 2 TCX proteins represented in both A. thaliana and O. sativa are also found in legumes (Cvitanich et al., 2000) and appear to be ubiquitous in flowering plants, both monocots and dicots. The further expansion of the TCX protein family in rice could indicate that plant TCX proteins have been evolving rapidly—the relatively large number of duplications allowing different members to take on new functions. It also appears that a proportion of the plant TCX genes could be turning into pseudogenes. Prominent examples could be the AtTCX7 and -8 genes, for which it was not possible to detect expression and which do not contain the normal complement of type 2 conserved sequences.
Mutations in the AtTSO1 gene disrupt development of both male and female reproductive tissue
The phenotypes of the tso1-5 and tso1-6 mutants characterized here differ from those of previously described tso1 mutants. In both tso1-5 and tso1-6 (Fig. 5B, C), sepals, petals, stamens, and carpels are all clearly distinguishable, in contrast to the severe tso1-1 mutant (Liu et al., 1997; Song et al., 2000). On the other hand, phenotypic aberrations are not restricted to ovules as in the mild tso1-3 allele (Hauser et al., 1998, 2000). In the male part of the flower, anthers from both tso1-5 and tso1-6 mutants contained a mixture of enlarged and collapsed pollen grains. Similar defects have been reported for gsl1 gsl5 callose synthase double mutants, but no defects in ovule or carpel development were described (Enns et al., 2005). Incomplete fusion of the carpels reminiscent of that seen in tso1-6 mutants has been observed in Arabidopsis mutants such as spatula, crabs claw, and ettin (Sessions et al., 1997; Alvarez and Smyth, 1999; Bowman and Smyth, 1999; Heisler et al., 2001). None of these mutations, however, affect ovule and pollen phenotype, and the tso1-5 and tso1-6 mutants described here seem to be unique in disrupting the development of both male and female reproductive tissue by causing aberrant cell shapes and preventing proper organ fusion.
Phenotypic differences between tso1 mutants could be explained by an overlap in AtTSO1 and AtTCX2 function
Three types of tso1 mutants have been described so far. The strongest alleles, tso1-1 and tso1-2, have cysteine to tyrosine transitions in the CRC domain and cause severe alterations in floral organ shape and inflorescence meristem organization as well as cell division defects (Hauser et al., 2000; Song et al., 2000). The tso1-3 and tso1-4 premature stop codon mutants are the weakest alleles, which specifically affect ovule development (Hauser et al., 1998). Finally, the tso1-5 and tso1-6 T-DNA insertion mutants described here represent intermediate alleles where morphological changes were restricted to reproductive tissues but where pollen, ovule, and carpel development were all influenced. The mutations in the weaker alleles, tso1-3, 4, 5, and 6, are located close to each other in the DNA sequence but result in putative protein products including different sets of conserved domains (Fig. 1E). Considering the phenotypic variation seen, some of the alleles could produce partially active TSO1 proteins, and the activity of these proteins would vary with their length. This is consistent with the detection of transcripts 5' of the insertion site in tso1-5 and tso1-6 mutants. It is striking that the loss of full-length proteins in the weaker alleles has such mild phenotypic effects compared with the single amino acid changes of the tso1-1 and tso1-2 alleles. This can now be explained in terms of the zinc binding by the CRC domain, since substitution of a single cysteine residue will most probably disturb co-ordination of zinc ions and lead to an altered protein structure.
The question remains, though, why the range of afflicted tissues differs between the mutants. One possibility is that proteins with functions overlapping that of AtTSO1 exist. They would be able to compensate for loss of AtTSO1 function where available at sufficient levels but be sensitive to the presence of modified full-length AtTSO1 protein. The present phylogenetic analysis has shown that AtTSO1 belongs to the plant-specific group of type 1 TCX proteins, which comprises AtTSO1, AtTCX2, AtTCX3, and AtTCX4 in A. thaliana. AtTCX4 lacks the GRK domain present in most type 1 TCX proteins and its expression could not be detected. The AtTCX3 gene is highly expressed in pollen, but is unable to compensate for loss of AtTSO1 function in this tissue, as demonstrated by the tso1-5 and tso1-6 mutants. This leaves AtTCX2 as the best candidate for having a functional overlap with AtTSO1 and indeed AtTCX2 has the most similar overall expression pattern to AtTSO1. However, AtTCX2 does not share the pollen expression with AtTSO1 and is expressed at lower levels in stage 15 carpels than AtTSO1. If AtTSO1 and AtTCX2 functions overlap, these tissues where AtTSO1 is expressed and AtTCX2 is absent or expressed at low levels would be predicted to be susceptible to loss of AtTSO1 function. In agreement with this, defects in carpel, ovule, and pollen development were observed in the tso1-5 and tso1-6 mutants described here. In the shoot apex, AtTSO1 and AtTCX2 are both expressed. However, as the vegetative meristem makes the transition to the inflorescence meristem, the AtTSO1/AtTCX2 expression ratio becomes higher, presumably making the inflorescence meristem more sensitive to the effects of a modified TSO1 protein, which agrees with the effects seen in the tso1-1 and tso1-2 mutants. Therefore, the zinc binding of the CRC domain and an overlap in function between AtTSO1 and AtTCX2 could explain the range of phenotypes seen in tso1 mutants.
Similarities between plant and animal TCX protein function
In A. thaliana, investigation of tso1 mutants has revealed that a plant TCX protein plays a role in development of both male and female reproductive tissues. Similar effects have been reported in animals, where loss of TCX function leads to developmental defects in C. elegans reproductive tissue, and mutations in the dREAM complex member dE2F2 cause defects in the fertility of both male and female D. melanogaster flies (Cayirlioglu et al., 2001; Frolov et al., 2001; Owen et al., 2003; Korenjak et al., 2004). On the mechanistic level, it has been shown that TCX and RB protein-containing dREAM complexes in D. melanogaster regulate sex- and developmental-specific genes in a TCX-dependent way (Korenjak et al., 2004). In addition, the H. sapiens HsTCX1 (hMip120) protein is able to bind RB in vitro, indicating a conservation of TCX–RB interaction and function throughout the animal kingdom (Korenjak et al., 2004). The phylogenetic distribution of TCX, RB, and E2F proteins also lends support to an extensive conservation of TCX function as RB and E2F proteins are present in all organisms in which TCX proteins were identified (not shown), while they are absent from prokaryotes and fungi (Lipsick, 2004). To determine if the direct RB–TCX protein interaction seen in animals is conserved in plants, a yeast two-hybrid direct interaction test was performed with AtTSO1 and AtRB-related protein. No interaction could be detected in this assay. However, considering the striking conservation of the CRC domain and the fact that TCX proteins have been associated with reproductive tissues in all organisms where they have been investigated, it is reasonable to suggest that plant TCX proteins, like their animal counterparts, could be part of chromatin-binding protein complexes that regulate sex-specific genes.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data are available at JXB online.
Table S1. Pearson correlation coefficients for pairs of TCX genes.
Table S2. Primers used in RT-PCR analysis.
Table S3. Primers used for insertion mutant genotyping.
Table S4. Experimental slides in the NASC Affymetrix database (Craigon et al., 2004), showing >2-fold the average TCX gene expression.
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Acknowledgements |
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We would like to acknowledge all authors who have published data in the NASC Affymetrix database. Without their contributions this study would not have been feasible. Thanks are also due to Dr Jan Trige Rasmussen, Margit Skriver Rasmussen, and Dr Ray Brown, Department of Molecular Biology, University of Aarhus, Denmark for assistance with the protein work. This work was supported by the Danish Biotechnology Programme and the EU TMR ERBFMRXCT980243 contract, and in part by funding from the UK Biotechnology and Biological Research Council and from the Grant Agency of the ASCR (grant B6038409). LS is supported by Danish Research Council grant SNF 21-01-0329.
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* Present address: Max Planck Institute for Developmental Biology, Spemannstrasse 37–39, D-72076 Tübingen, Germany.
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Abbreviations |
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AGI, Arabidopsis Genome Initiative; APT, adenine phosphoribosyltransferase; BLAST, basic local alignment search tool; CRC, cysteine-rich domain found in all TCX proteins; CXC, cysteine-rich motif found in the CRC domain; dREAM, Drosophila RB, E2F, and Myb; E(z), Enhancer of zeste; EST, expressed sequence tag; GST, glutathione S-transferase; GUS, β-glucuronidase; KAPP, kinase-associated protein phosphatase, U09505; NASC, Nottingham Arabidopsis Stock Centre; RB, retinoblastoma; RT-PCR, reverse transcriptase polymerase chain reaction; SynMuv, Synthetic Multivulva; TCX, tesmin/TSO1-like CXC; tso, Chinese for ‘ugly’.
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