Cloning of a wheat cDNA encoding an ortholog of the maize LIGULELESS4 homeobox protein
Yoshihito Ishida* and Shigeo Takumi
Laboratory of Plant Genetics, Graduate School of Agricultural Science, Kobe University, Nada-ku, Kobe 657-8501, Japan
*present address: Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8397, Japan
corresponding author: Shigeo Takumi
E-mail: takumi@kobe-u.ac.jp
The shoot apical meristem (SAM) plays a central role in plant development, determination of cell fate and differentiation of vegetative tissues. Class I Knotted1-like homeobox (KNOX) genes maintain the indeterminancy of SAM and act in subsequent shoot development (Kerstetter et al. 1997; Sentoku et al. 1999). A maize semidominant mutation Liguleless3 (Lg3) allele disrupts the leaf at the ligular region of the midrib by transforming blade, auricle and ligule to sheath-like tissue (Fowler and Freeling 1996). The lg3 gene encodes a homeodomain protein in the KNOX family (Muehlbauer et al. 1999). Other dominant leaf mutants such as Knotted1 (Kn1), Rough sheath1 (Rs1) and Liguleless4 (Lg4) exhibit similar blade-to-sheath transformations in maize (Freeling and Hake 1985; Becraft and Freeling 1994; Fowler and Freeling 1996), and the kn1, rs1 and lg4 genes also belong to the KNOX family (Kerstetter et al. 1994; Bauer et al. 2004). Two KNOX genes, Wknox1 and WRS1, were isolated in common wheat (Takumi et al. 2000; Morimoto et al. 2005, 2009). The previous expression analyses and transgenic studies indicated that Wknox1 and WRS1 are functionally orthologous to kn1 and rs1, respectively. Here, we reported cloning of a cDNA encoding an lg4 ortholog from young spikes of common wheat.
Partial cDNA sequences of wheat KNOX family were previously reported (Takumi et al. 2000). Based on one of the partial sequences, the cDNA-specific primer was designed for 5'- and 3'-RACE PCR to isolate a full-length cDNA sequence. The RACE PCR was performed using mRNA isolated from young spikes of a common wheat (Triticum aestivum L.) cultivar 'Chinese Spring' (CS) and SMART RACE cDNA Amplification Kit (Clontech). After sequencing of the RACE PCR products, a full-length cDNA fragment was isolated (Fig. 1).
This cDNA contained a complete open reading frame encoding putatively a KN1-type homeobox protein with 306 amino acid residues that showed amino acid identities of 75.8% with rice OSH71, 73.8% with maize LG4b and 70.9% with maize LG4a (Fig. 2). lg4a (formerly knox11) and lg4b (knox5) are likely to encode the redundant function (Bauer et al. 2004), and closely linked duplicates on maize chromosome 8L (Kerstetter et al. 1994). OSH71 is a rice ortholog of maize lg4 genes (Sentoku et al. 1999). The high amino acid identities were observed in three conserved domains of the KN1-type homeobox proteins, KNOX, ELK and Homeo domains, among the isolated cDNA-encoding protein, OSH71 and LG4. Our identified cDNA-encoding protein was phylogenetically closer to the LG4 and OSH71 proteins rather than their closely related LG3 and OSH6 (Fig. 3). Therefore, we named the lg4-like cDNA as wheat LIGULELESS4 (WLG4). WLG4 cDNA sequence was deposited in the DDBJ database under the accession number AB465042.
To study the copy number of WLG4 in the wheat genome, Southern blots were analyzed using total DNA isolated from diploid (T. monococcum), tetraploid (T. durum cv. 'Langdon') and hexaploid wheat (CS). Southern blots showed three, two and one copy numbers of WLG4 in the hexaploid, tetraploid and diploid wheat genomes, respectively (data not shown). The results indicated that the common wheat genome possessed three homoeologous loci of the WLG4 gene, one in each of the three components, A, B, and D genomes.
To study the expression pattern of WLG4 in various organs of wheat, RT-PCR analysis was performed. WLG4 transcripts were abundantly accumulated in SAM-containing embryos and young spikes, and floral organs, but not in leaf blade, sheath and ligule/auricle (Fig. 4). Maize lg3 and lg4 mRNA is expressed in apical regions but is not expressed in leaves of wild-type plants (Muehlbauer et al. 1999). Detail in situ hybridization analysis demonstrated that the expression pattern of rice OSH71 is similar to that of OSH6, a rice lg3 ortholog, and that the expression of OSH6 and OSH71 is downregulated in the SAM and in turn is localized at the boundaries of the shoot lateral organs (Sentoku et al. 1999). WLG4 expression pattern revealed by RT-PCR analysis was corresponding to the previously reported results in rice and maize. Detail expression studies should be required to elucidate the WLG4 function in SAM and lateral organ development of wheat.
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