JXB Advance Access originally published online on April 18, 2005
Journal of Experimental Botany 2005 56(416):1605-1614; doi:10.1093/jxb/eri155
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Expression patterns of TEL genes in Poaceae suggest a conserved association with cell differentiation
1Institut de Biotechnologie des Plantes (UMR8618), University of Paris XI, F-91405 Orsay Cedex, France
2INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, F-78026 Versailles Cedex, France
* To whom correspondence should be addressed. Fax: +33 1 69 15 34 24. E-mail: charon{at}ibp.u-psud.fr
Received 4 October 2004; Accepted 1 March 2005
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Abstract |
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Poaceae species present a conserved distichous phyllotaxy (leaf position along the stem) and share common properties with respect to leaf initiation. The goal of this work was to determine if these common traits imply common genes. Therefore, homologues of the maize TERMINAL EAR1 gene in Poaceae were studied. This gene encodes an RNA-binding motif (RRM) protein, that is suggested to regulate leaf initiation. Using degenerate primers, one unique tel (terminal ear1-like) gene from seven Poaceae members, covering almost all the phylogenetic tree of the family, was identified by PCR. These genes present a very high degree of similarity, a much conserved exon–intron structure, and the three RRMs and TEL characteristic motifs. The evolution of tel sequences in Poaceae strongly correlates with the known phylogenetic tree of this family. RT-PCR gene expression analyses show conserved tel expression in the shoot apex in all species, suggesting functional orthology between these genes. In addition, in situ hybridization experiments with specific antisense probes show tel transcript accumulation in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells. Tel expression is not restricted to initiating leaves as it is also found in pro-vascular tissues, root meristems, and immature inflorescences. Therefore, these results suggest that TEL is not only associated with leaf initiation but more generally with cell differentiation in Poaceae.
Key words: Cell differentiation, evolution, Poaceae, terminal ear1
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Introduction |
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Higher plants, unlike animals, elaborate much of their architecture post-embryonically through meristems, which are groups of pluripotent stem cells localized at the tips of the embryo shoot–root axis. Meristems consist of well-organized regions, able by division and differentiation to initiate tissues and new organs and to maintain themselves. In contrast to the root apical meristem (RAM), which only participates in root tissue formation, the shoot apical meristem (SAM) not only produces stem tissues, but also generates leaves, buds, and flowers. All these processes imply cell division and cell differentiation. For example, leaf initiation at the flanks of the SAM by groups of cells recruited for and called leaf founder cells, involves co-ordinated changes in the polarity and the rate of cell division and expansion (Smith and Hake, 1992
).
In grasses, such as rice and maize, the recruitment of leaf founder cells begins at a single site on the flank of the SAM and continues laterally around the circumference of the apex, until a ring-like domain on the periphery of the SAM is formed. In maize, fate mapping analyses demonstrated that approximately 200 founder cells are recruited from the SAM to become a leaf (Poethig, 1984). Then, development of the leaf occurs basipetally, such that the apical domains of the upper leaf blade differentiate first, and the lower domains of the leaf sheath differentiate last (Poethig and Szymkowiak, 1995; Scanlon, 2003).
The acquisition of founder cell identity appears to be determined by the knox (knotted1-like homeobox) genes which, in many species, are expressed in the meristem, but not in founder cells and lateral organ primordia (Smith et al., 1992; Jackson et al., 1994; Schneeberger et al., 1995; Scanlon et al., 1996). A notable exception is in tomato, where the LeT6 knox gene was also found in the leaf primordia of compound leaves (Kim et al., 2003). The Arabidopsis knox gene SHOOT MERISTEMLESS (stm) maintains stem cell fate by negative regulation of the myb domain transcription factor ASYMMETRIC LEAVES1 (as1) and a member of the LOB-like transcription factor family ASYMMETRIC LEAVES2 (as2) (Byrne et al., 2000, 2002; Iwakawa et al., 2002; Shuai et al., 2002). Conversely, the rs2/phan/as1 genes are expressed in lateral organ primordia where they function as negative regulators of knox genes (Schneeberger et al., 1998; Waites et al., 1998; Timmermans et al., 1999; Tsiantis et al., 1999; Byrne et al., 2000; Ori et al., 2000; Semiarti et al., 2001, Sun et al., 2002).
The terminal ear1 gene was also proposed to play a role in the regulation of leaf initiation in maize (Veit et al., 1998). Indeed, te1 maize mutants, first named for the ear-like inflorescence that forms in place of the normal terminal tassel (Matthews et al., 1974), present tassel feminization, but also abnormally short internodes, and abnormal leaf initiation and development (Veit et al., 1993, 1998). Interestingly, te1 mutants exhibit abnormally positioned leaf primordia, although normal down-regulation of KNOTTED1 transcripts is observed (Veit et al., 1998). The Zmte1 gene was shown to be expressed in semi-circular bands in the shoot apex, excluding the site of organ initiation, suggesting that Zmte1 could inhibit lateral organ initiation and differentiation (Veit et al., 1998). Zmte1 belongs to the multigenic mei2-like gene family (Anderson et al., 2004; Jeffares et al., 2004). Mei2 was first isolated from Schizosaccharomyces pombe (Watanabe and Yamamoto, 1994), and shown to be required for both pre-meiotic DNA synthesis and the first meiotic division (meiosis I). Both proteins, ZmTE1 and MEI2, contain three conserved RNA-recognition motifs (RRMs) indicating that TE1 may function through an RNA-binding activity (Veit et al., 1998; Anderson et al., 2004; Jeffares et al., 2004). Phylogenetic analyses in the Zea genus revealed a neutral pattern of evolution for TERMINAL EAR1 (White and Doebley, 1999), suggesting that TE1 has a basic role in plant development.
The two model systems, maize (Zea mays) and rice (Oryza sativa), belong to the grasses (Poaceae), an important group of monocots. Recent phylogenetic studies have led taxonomists to recognize four major lineages within the grasses: the Pooideae (wheat, barley, oats, and rye), the Oryzoideae (rice), the Panicoideae (maize, sorghum, hairy crabgrass, and pearl millet) and the Chloridoideae (finger millet and tef) (Kellogg, 1998; Laurie and Devos, 2002; Fig. 1). Members of this family present great physiological, morphological, and genetic diversity (Kellogg, 1998), but they share a number of common properties with respect to the leaf initiation process (recruitment of leaf founder cells at one site of the SAM, development of the leaf primordium until forming a ring-like domain at the periphery of the SAM, basipetal development of the leaf, and distichous leaf position) (Steeves and Sussex, 1989; Scanlon, 2003). The goal of this work was to determine if these common traits imply common genes, like te1.
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Taking advantage of the rice genome sequence (Goff et al., 2002
) and maize genomic data (Veit et al., 1998), one single tel (terminal ear1-like) gene was identified from all the seven selected Poaceae species by PCR using degenerate primers. This paper presents the characterization of these genes and their phylogenetic relationships, and describes their expression patterns. In addition, a more detailed picture of tel gene expression in the different species studied leads to the postulation that TEL is not only associated with leaf initiation but more generally to cell differentiation in the Poaceae. |
Materials and methods |
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Nomenclature
Italicized names refer to genes and gene fragments (e.g. tel), whereas non-italic upper case names (e.g. TEL) refer to predicted protein.
Plant material
Different species of grasses (Poaceae family) were used for this study: rice (Oryza sativa ssp. japonica cv. Nipponbare), maize (Zea mays cv. W22), sorghum (Sorghum bicolor cv. Tamara), hairy crabgrass (Digitaria sanguinalis), barley (Hordeum vulgare cv. IGRI), rye (Secale cereale), pearl millet (Pennisetum glaucum cv. 25.95), and xTriticosecale. Seeds were germinated in vermiculite and grown in the greenhouse at 24 °C under a 16 h light period.
DNA extraction
Genomic DNA was extracted from fresh or frozen leaf material following a modified version of the CTAB procedure of Doyle and Doyle (1987). Plant tissues were ground in liquid nitrogen, transferred to a microcentrifuge tube and resuspended in extraction buffer (2% (w/v) CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM TRIS–HCl, and 0.2% ß-mercaptoethanol, pH 8). Samples were incubated at 60 °C for 30 min with occasional mixing. Cooled samples were then extracted with phenol:chloroform (1:1, v/v) followed by chloroform, precipitated with isopropanol, washed, dried, and resuspended in 100 µl of distilled water. RNA was removed using RNase A treatment according to the manufacturer (GibcoBRL), and DNA was further purified by ethanol precipitation.
Cloning and sequencing of Poaceae tel genes
Two spanning regions, one of 1.14 kilobases (kb) in 5' and one of 1.5 kb in 3' of the putative gene (covering almost all the tel gene), were amplified from total DNA of selected Poaceae species using degenerate Poa primers. For the 5' amplification, the forward Poa12 primer (5'-GCTTCCGCAMCAGCTDTACTGCC-3') was used with the reverse Poa5 primer (5'-GCACCACKCCTTCCCTCGCC-3'). For the 3' amplification, the forward Poa5bis primer (5'-GGCGAGGGAAGGMGTGGTGC-3') was used with the reverse Poa1 primer (5'-CCACGGGCAGGTACTCGTCG-3'). For Pooideae species, the forward Poa8 primer (5'-GTCCGCGAGCAGCACATSCG-3') was used with the reverse Poa13 primer (5'-GTTGAGCAGCAGCTTCTGGC-3') for the 5' amplification, and the forward Poa14 primer (5'-GCTCAACATGCTGGACAACC-3') with the Poa1 primer (see above) for the 3' amplification. These primers were designed from sequence comparison between te1 from maize (Zmte1, AF348319
[GenBank]
) and te1-like from rice (Ostel1, AP003380
[GenBank]
.3). PCR amplifications were carried out in a 50 µl mix containing 1.5 mM MgCl2, 10 pM dNTP mix, 50 pM of each primer (MWG Biotech), 10 µl of Q solution (Qiagen), 1 unit of Taq DNA polymerase (Qiagen), and 100–200 ng of DNA. The thermocycling conditions consisted of an initial denaturation step at 95 °C (3 min), followed by 30 cycles of denaturation at 94 °C (1 min), annealing at 55–60 °C (1 min), and extension at 72 °C (1.5 min), followed by a final extension step at 72 °C (5 min). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and cloned using pGEM-T Easy Cloning Kit (Promega) for sequencing. Dye-terminator cycle sequencing was carried out (Applied Biosystems) and samples were analysed on an ABI 377 Prism Automatic DNA Sequencer (Applied Biosystems). For each species and amplified region, 10–20 clones were sequenced from 2–3 independent PCR reactions.
RNA extraction and RT-PCR experiments
Total RNA was prepared using the guanidinium chloride method (Logemann et al., 1987) and DNase-treated according to the manufacturer (Promega). Semi-quantitative RT-PCR studies were performed using total RNA from various plant tissues. Briefly first-strand cDNA synthesis was carried out with the Superscript II Reverse Transcriptase (Invitrogen, Life Technologies) and a poly (dT) primer (Promega). Ten per cent of the first-strand cDNA reaction mix was used in a 50 µl PCR reaction containing 1.5 mM MgCl2, 10 pM dNTP mix, 50 pM of each primer (MWG Biotech), 10 µl of Q solution (Qiagen), and 2.5 units of Taq polymerase (Qiagen). The thermocycling conditions were as follows: 3 min at 95 °C, 30 (actin gene) or 35 (tel gene) cycles of 1 min at 94 °C, 1 min at 52 °C (actin) or 55 °C (tel) and 1.5 min at 72 °C, and a final extension of 5 min at 72 °C. To control for a difference in loading, the actin gene was amplified from the same cDNA preparation. The PCR primers used were the following: actin, forward primer 5'-AACTGGGATGATATGGAGAA-3' and reverse primer 5'-CCTCCAATCCAGACACTGTA-3'; Zmte1: Poa12 and Poa5 primers (see above); Ostel1: 5'-GCTGGACAACCACTGCATCC-3' and 5'-GGATCCGTAGCCACCATCATTGCC-3'; other tel genes: Poa5bis and Poa1 primers or Poa14 and Poa1 (see above). RT-PCR products were separated on a 1% agarose gel. The expected fragments were purified using QIAquick Gel Extraction Kit (Qiagen), cloned into pGEM-T Easy Vector (Promega) and confirmed by sequence analysis (as above).
In situ hybridization
In situ hybridizations were performed mainly as described in Laufs et al. (1998). Briefly plant tissues were fixed in FAA solution (McKhann and Hirsch, 1993) under vacuum for 30 min, and left in fixative overnight at 4°C. After fixation, tissues were washed in PBS, dehydrated through a graded ethanol series, and embedded in Paraplast Plus (Sherwood Medical), essentially as described by Jackson (1991). Microtome sections (8–10 µm thick) were applied to precoated glass slides (DAKO). Antisense probes were synthesized with digoxigenin (DIG-UTP, Roche Diagnostics) using the Riboprobe In Vitro Transcription System (Promega). Immunodetection of the DIG-labelled probes was performed using an anti-DIG antibody coupled to alkaline phosphatase (Roche Diagnostics). The following probes were used: cDNA fragments for Ostel1, Sbtel1, and Dstel1 (RT-PCR fragment), DNA fragments for Zmte1, Sctel1, and Hvtel1.
Phylogenetic analysis
Sequences were aligned with Clustal X (Thompson et al., 1997), followed by manual adjustments using BIOEDIT. Phylogenetic trees were constructed with the Neighbor–Joining algorithm using the NEIGHBOR program of the PHYLIP package (Felsenstein, 1993). Evolutionary distances were estimated by the PHYLIP program DNADIS under the Kimura and Jukes–Cantor matrices. To test the statistical significance of the phylogenetic trees, 100 bootstrap samples were generated from each data set using the SEQBOOT program of the PHYLIP package, and a consensus tree was produced by CONSENSE. Phylogenetic trees were also inferred by the Bayesian methods, using MrBayes v3.0 (Huelsenbeck and Ronquist, 2001). One cold and three incrementally heated chains were run for 2 000 000 generations, with random starting trees and a temperature value of 0.2. Trees were sampled every 100 generations. The first 10 000 trees, corresponding to samples made before the chain has reached a stable state, were discarded. 10 000 trees were sampled for inferring a Bayesian tree. Trees were visualized using TreeView program (Page, 1996).
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Results |
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Identification of tel genes in Poaceae
In order to identify tel genes from Poaceae, a homology search in databanks followed by alignment using the Zmte1 gene sequence were first performed. One complete gene sequence was identified in rice and called Ostel1 gene (previously named OML1, Jeffares et al., 2004
) (Table 1). Other rice sequences were found but they only corresponded to small regions (50–150 nucleotides) of the Zmte1 gene, spread in the entire rice genome and, for that reason, were left out. A BLASTN search at the NCBI was also performed against Poaceae EST databanks. Not surprisingly, three ESTs from maize were found as well as one EST from barley with E values below 2 e-65 (score >250) and 1 from sugarcane with an E value below 6 e-51 (score=531) (Table 1), confirming the existence and the expression of tel genes in other Poaceae species.
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Then, the alignment of the two complete Zmte1 and Ostel1 genes was used to draw degenerate primers (called Poa primers), in order to search for tel sequences in other Poaceae species by a PCR strategy on genomic DNA. To avoid the amplification of non tel-related RRM genes, only primers drawn outside the RRM regions (RNA-recognition motifs) were used at first. Two sets of PCR were performed (with Poa12 and Poa5, Poa5bis and Poa1 primers, see Materials and methods), spanning almost all the predicted tel genes (Fig. 2A). Amplified fragments were subcloned into a pGEM-T easy vector (Promega) and more than 10 independent clones per species were randomly selected for sequencing. For rye and barley, these PCR amplifications led to non-tel-related retrotransposon sequences due to the use of Poa5 and Poa5bis (N Paquet et al., unpublished data). Consequently, two new sets of primers were designed and used ([Poa8–Poa13] and [Poa14–Poa1], Fig. 2A), taking advantage of nucleotide conservation in the RRM3 region. As above, PCR was performed; amplified fragments were subcloned and finally sequenced.
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Sequence analysis revealed one tel gene in all the selected species (Table 1). One unique tel sequence was found in sorghum, hairy crabgrass, barley, rye, and pearl millet, as was found in rice. Identification of tel sequences in hexaploid species (as xTriticosecale) led to three different tel genes, confirming the existence of one tel copy per genome.
Sequence analysis of Poaceae tel genes
Multiple sequence alignments were performed using Clustal X. Sequence identity among all the identified tel genes at the DNA level ranges from 60% to 98% in the [Poa8–Poa13] region, with the highest score between rye and xTriticosecale (98%) and the lowest between sorghum and xTriticosecale (60%). In the [Poa14–Poa1] region, sequence identity ranges from 85% (between rye and maize) to 99% (between barley and sorghum) at the DNA level. But this high identity level was probably due to the fact that this [Poa14–Poa1] region spans part of the third putative RNA-binding motif (Fig. 2A), and not because of a divergence between 5' and 3' genomic identity. Alignment of peptide sequences derived from these two regions ([Poa8–Poa13] and [Poa14–Poa1] with the exception of primers) showed identical results, suggesting a high sequence conservation between all the Poaceae identified tel genes.
Detailed sequence analysis revealed that all the Poaceae tel genes share an identical structure as shown by the existence of five size-comparable introns located at identical positions (Fig. 2A), as it was further confirmed by sequencing RT-PCR products. Moreover, at the amino acid level, the three RNA-binding motifs previously characterized in ZmTE1, were recovered in all the tel genes, as well as the TEL motif (Fig. 2B), a characteristic insertion found in the third RRM and allowing discrimination between Zmte1-like and other mei2-like genes (Anderson et al., 2004; Jeffares et al., 2004; N Paquet, unpublished data). Multiple RRM motif alignments from the Poaceae tel genes are shown in Fig. 2B. Even though a RRM1 sequence was not available for rye and barley, these alignments reveal the very high sequence conservation within each of the three RRMs, especially for the third RRM in which 100% identity was found for the 26 amino acids just localized upstream the TEL motif (Fig. 2B). These observations suggest a relatively low selection pressure within these domains compared with the entire TEL proteins and, moreover, a strong selection pressure within the third RRM, probably indicating RRM3 importance in TEL function.
In order to evaluate tel gene evolution within Poaceae, phylogenetic trees were constructed from nucleotide sequence comparison of the Poaceae TEL proteins. Different phylogenetic methods were used (Neighbour–Joining algorithm and Bayesian inference) and showed identical results (Fig. 3). It appears that Poaceae tel genes have evolved according to the phylogenetic tree of the family (Fig. 1). This is consistent with the hypothesis of a conserved function.
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Expression studies of Poaceae tel genes
In order to characterize the tissue-specific expression pattern of Poaceae tel genes, RNA was isolated from roots, leaves, and apex (the shoot apical meristem surrounded by youngest leaves) of 3–4 leaf-stage plantlets. Semi-quantitative RT-PCR analyses were performed using different pairs of Poa primers (Fig. 4). Each resulting amplification product was cloned and sequenced to confirm the tel specificity and predicted intron–exon structure. The actin gene was used as a control for RNA extraction and RT-PCR experiments. In all the tested species, tel was expressed in the apex region (Fig. 4). Surprisingly, in hairy crabgrass and sorghum, expression was also detected in leaves and roots, but with a lower signal (Fig. 4D, E).
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The expression pattern of tel genes was further characterized by in situ hybridization using DIG-UTP-labelled RNA probes. Figure 5 shows a detailed analysis in Digitaria sanguinalis. During vegetative development of this species, tel transcripts were found in all leaf founder cells and leaf primordia (Fig. 5A, B). In later stages of leaf development, tel expression became restricted to the major vascular bundles and leaf margins (Fig. 5C). No expression was found in the central quiescent zone of the shoot apical meristem nor in the internode zone, except in pro-vascular tissues connecting young leaves to the stem, where a weak expression could be detected (Fig. 5A). Surprisingly, strong signals were also found in adventitious root apices, more precisely in all differentiating tissues of the root apical meristem (initials of epidermis and cortex, pro-vascular tissues, initials of root cap, and columella), but not in the quiescent centre (Fig. 5D). A tel sense probe used as a negative control did not show any signal in shoot or root apex tissues (data not shown).
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Identical tel expression analyses by in situ hybridization were performed for all the selected species, and revealed similar expression pattern (Fig. 6, data not shown). Indeed, especially during leaf initiation and development, tel transcripts were found in all leaf founder cells, leaf primordia (Fig. 6A, C, G), and then restricted to leaf margins (Fig. 6E). Tel expression in leaf margins was particularly well observed by in situ hybridization of shoot apex transversal sections (Fig. 6B, D, F, H). When axillary buds and immature inflorescence were present in the sections, strong signals were also detected in these tissues, especially for maize, rice, and hairy crabgrass (Fig. 5A; data not shown). These results suggest that tel expression is not restricted to the vegetative development of the shoot apex, but is also expressed in pro-vascular tissues, root apice, and immature inflorescences.
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Because these expression results in the shoot apex (or at least the interpretation of these results) were in slight contradiction with previously published studies of Zmte1 expression (Veit et al., 1998
), tel expression was further analysed in detail during leaf initiation by in situ hybridization (Fig. 7). Transverse serial sections of different Poaceae shoot apices (especially in maize) revealed that tel transcripts could be detected in all leaf founder cells until the margins of the primordium meet together (data not shown). Then, during leaf development, the expression of tel could be found in specific (and more and more restricted) zones of the leaf primordium: at its apical part (P1 primordium, Fig. 7A), and in its right and left margin domains until the complete leaf development (Fig. 7B–D). These results show that tel is not expressed in the internode zone of the stem, but in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells (Fig. 7E, F).
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Discussion |
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The evolution of tel genes
In the present study, the possible existence and expression pattern of TERMINAL EAR1 homologues in Poaceae was investigated, a gene previously identified in maize and suggested to regulate leaf initiation (Veit et al., 1998
). One tel (terminal ear1-like) gene has been identified in each of the seven Poaceae species tested. Sequence analyses showed that these genes share a very high degree of similarity, a much conserved exon/intron structure, and identical expression pattern, suggesting not only a conservation of molecular structure, but also a functional orthology link. To confirm functional conservation, the characterization of tel rice mutants, cross-complementation of the te1 maize mutant by Ostel1, as well as synteny analysis, would be required. To confirm the existence of one single tel gene per genome, a hexaploid Poaceae species (xTriticosecale, AABBRR genome) was integrated in this analysis, which led to the identification of three different tel sequences. Phylogenetic studies revealed that one xTriticosecale sequence (Ttel1) is closer to ScTel1 than the two other ones (Ttel2 and Ttel3, Fig. 3). Moreover, the existence of short arms in the tree between Ttel1 and Sctel1 (Fig. 3), suggests a very recent divergence. These results are in agreement with the fact that xTriticosecale results from a recent human-made-cross between Secale cereale (diploid species, RR genome) and Triticum durum (tetraploid species, AABB genome), and the theory of one expected sequence from the RR genome and two from the AABB genome. Looking for tel genes in T. durum would confirm this hypothesis.
It was previously shown that te1 presents a neutral pattern of evolution in the Zea genus (White and Doebley, 1999). Here, phylogenetic analyses also suggest a neutral evolution of the tel gene in Poaceae, although more species should be analysed (domesticated and non-domesticated species) to strengthen this hypothesis. A neutral pattern of evolution was also proposed for other genes implicated in architecture control, suggesting that morphological variation may depend more on the differences in the expression patterns of genes (Doebley and Lukens, 1998) than on diversifying genes. As a consequence, promoter and regulating sequences should also be studied in order to analyse morphological evolution. In the case of tel even the regulation of expression seems to be highly conserved, since identical expression patterns were found for all the Poaceae tel genes. The comparison of their promoters and regulating sequences would allow the identification of particular motifs or domains implicated in this process.
Expression patterns of tel genes in Poaceae and putative functions
Leaf initiation starts at the SAM by the recruitment of leaf founder cells. Although many hypotheses have been proposed to explain how the plant spatially and temporally regulates this recruitment (Reinhardt et al., 2003), the implicated mechanisms remain unclear. Due to its expression patterns in maize, TERMINAL EAR1 was suggested to act as a negative regulator of leaf initiation (Veit et al., 1998). In the present paper, the data confirmed the fact that te1 and more generally all the tel genes identified in this work have an expression related to leaf initiation and development. Nevertheless, as clearly observed using in situ hybridization on serial sections of vegetative shoot apex, tel is not expressed in the internode zone of the stem as it was previously published (Veit et al., 1998), but is expressed in all differentiating cells of the leaf, from the recruitment of leaf founder cells to leaf margins cells (Figs 5–7
). As a consequence, tel appears not to be a negative but a positive regulator of leaf initiation. This new interpretation is still consistent with Zmte1 mutant analysis, where abnormally positioned leaf initiation was observed (Veit et al., 1998; N Paquet, unpublished observations). Expression data also fit well with the blade and midrib defects exhibited by the mutant, suggesting the importance of tel requirement in cell differentiation during plant leaf initiation and development.
ZmTE1 contains three conserved RNA-binding motifs (RRMs) and shows significant similarity to the Schizosaccharomyces pombe MEI2 protein (Veit et al., 1998; Anderson et al., 2004; Jeffares et al., 2004). Comparison of the Poaceae TEL sequences identified in this study confirmed the presence of the three RRMs in the TEL proteins of Poaceae. All these RRMs are much conserved, especially the third one which presents the previously identified TEL characteristic motif (Jeffares et al., 2004). This TEL motif appears to allow sequence and putative functional discrimination between tel and other mei2-like genes. As already shown for other RNA-binding proteins (Fedoroff, 2002), RNA interaction specificity depends on the sequence and the structure of the RRM. As a consequence, TEL may function through an RNA-binding activity partly divergent to MEI2 activity. The abundance of Arabidopsis thaliana genes encoding RNA-binding proteins (Lorkovic and Barta, 2002), and the recent reports implicating RNA-binding proteins in hormone signalling (Lu and Fedoroff, 2000; Hugouvieux et al., 2001; Xiong et al., 2001), suggest that such proteins emerge as important players in plant morphogenesis and cellular regulation (Burd and Dreyfuss, 1994; Fedoroff, 2002). MEI2 was shown to bind the MEI2-RNA and to relocalize this messenger to the nucleus (Ohno and Mattaj, 1999), as is the case for other RNA-binding proteins and corresponding RNA partners (Campalans et al., 2004). It would be interesting to investigate whether TEL proteins change their cellular localization during cell differentiation and identify their RNA partners.
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Acknowledgements |
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We gratefully acknowledge J de Buyser, J Vidal, and E Guiderdoni for Poaceae seeds. We also thank M-S Remigereau for the gift of the Pgtel1 sequence and pearl millet DNA, J Kronenberger for help and technical advice regarding in situ hybridization, L Garcia Haro for technical help, and Y Henry for useful discussion. This work was supported by a grant from the Ministère Français de l'Enseignement Supérieur et de la Recherche.
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