Comparison of three hemA1 cDNA sequences putatively encoding a glutamyl tRNA reductase in common wheat
Yu Takenouchi1, Kengo Kanamaru2 and Shigeo Takumi1
1: Laboratory of Plant Genetics, Graduate School of Agricultural Science, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan
2: Laboratory of Biological Chemistry, Graduate School of Agricultural Science, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan
Corresponding author: Shigeo Takumi
E-mail: takumi@kobe-u.ac.jp
Tetrapyrroles play important roles in various biological processes and inter-organellar signaling of higher plants (Tanaka and Tanaka 2007; Mochizuki et al. 2010). Four classes of tetrapyrroles including chlorophyll, heme, siroheme and pytochromobilin are found in higher plants, and a common biosynthetic pathway produces these tetrapyrroles mainly in plastids (Fig. 1). Glutamyl-tRNA syntherase ligates glutamate with tRNAGlu, and then glutamyl-tRNA reductase (GluTR) reduces the activated carboxyl group to produce glutamate-1-smialdehyde (GSA) (Tanaka and Tanaka 2007). Subsequently, GSA amino transferase (GSA-AT) converts GSA into 5-aminolevulinic acid (ALA). ALA is used as the universal precursor of tetrapyrrole biosynthesis in various organisms (Beale 1999). High concentration of ALA reduces the plant growth, whereas application of ALA at low concentrations enhances the growth, yield and salt tolerance (Hotta et al. 1997a, 1997b; Hara et al. 2011).
ALA formation is the first regulatory point in the tetrapyrrole biosynthetic pathway. Among the three steps of ALA synthesis, glutamyl-tRNA reduction is the most important one for the regulation (Goslings et al. 2004; Tanaka and Tanaka 2007). A previous study of the Arabidopsis fluorescent (flu) mutant elucidated that glutamyl-tRNA reduction is the limiting step of chlorophyll biosynthesis (Goslings et al. 2004). GluTR is encoded by the small hemA gene family in angiosperm (Tanaka and Tanaka 2007). Higher plants contain at least two hemA genes, and one of these genes, hemA1, is expressed primarily in photosynthetic tissues (Tanaka et al. 1996). The hemA1 expression is dependent on light and regulated by circadian rhythms (Matsumoto et al. 2004). Here, we report cloning of three homoeologous hemA1 (TaHEMA1) cDNAs from seedling leaves of common wheat (Triticum aestivum L.).
The hemA1 cDNA clones were isolated by reverse-transcription (RT)-PCR using total RNA from seedling leaves of common wheat cultivar Chinese Spring. Based on the nucleotide sequences of a barley hemA1 cDNA (D88382 and X92403; Bougri and Grimm 1996; Tanaka et al. 1997), a gene-specific primer set was designed as followed: 5’-ATGGCGGGAGCGACGTCAGC-3’ and 5’-TAGAGAAGACCCAAAGCTGA-3’. Amplified cDNAs of hemA1 were cloned into the pGEM-T vector (Promega, Madison, WI, USA) and nucleotide sequences were determined by an automated fluorescent Dye Deoxy terminator cycle sequencing system using an ABI PRISM 310 genetic analyser (PE Applied Biosystems, Foster City, CA, USA). Nucleotide sequences of the isolated cDNA clones and the predicted amino acid sequences were analyzed by GENETYX-MAC version 12.00 software (Whitehead Institute of Biomedical Research, Cambridge, MA, USA) and the sequence was searched for homology using the BLAST algorithm (Karlin and Altschul 1993).
Three cDNA sequences, TaHEMA1-1, TaHEMA1-2 and TaHEMA1-3, with a complete ORF were isolated from the seedling leaves. The cDNA sequences were deposited into the DDBJ database under these accession numbers: AB678198 (TaHEMA1-1), AB678199 (TaHEMA1-2) and AB678200 (TaHEMA1-3). The three TaHEMA1 cDNAs respectively contained single ORFs encoding 528, 527 and 528 amino acid residues, and showed high homology with the barley hemA1 cDNA. Comparison of the three TaHEMA1 cDNA sequences exhibited that nucleotide substitutions, insertions and deletions occurred within their open reading frames (ORFs), and that no frame-shift mutation was recognized (Fig. 2). The deletion mutations occurred in the 5’ region of ORF, in which a signal peptide for plastid-targeting was putatively encoded. More than 97% identities were found in putative amino acid sequences of the C-terminal enzymatic region in the three wheat GluTR proteins.
A phylogenetic tree of amino acid sequences of plant GluTR proteins was constructed by the unweighted pair group method with arithmetic mean (UPGMA) based on the Nei’s genetic distances. The tree clearly divided the plant GluTR sequences into monocotyledonous and dicotyledonous groups (Fig. 3). The barley and wheat GluTR sequences were closely related, and formed one subgroup in the monocotyledonous GluTR sequences. Therefore, the isolated TaHEMA1 cDNAs encoding GluTR seemed to be homoeologous in the common wheat genome.
ALA is metabotized by aminolevulinic acid dehydratase (ALAD) (Cornah et al. 2003). Three homoeologous ALAD1 (TaALAD1) cDNAs has been isolated from common wheat (Takenouchi et al. 2011). The wheat ALAD1 efficiently functioned to catalized from ALA to porphobilinogen (PBG) in transgenic tobacco plants. The TaALAD1 protein was localized in chloroplasts of guard cells in the epidermal cell layer revealed by transient expression of the TaALAD1-GFP fusion protein. Arabidopsis GluTR contains a plastid-targeting signal (McCormac et al. 2001), and the wheat GluTR is putatively localized in plastids. Wheat GluTR and ALAD1 cooperatively act in the tetrapyrrole biosysnthetic pathway in plastids.
In common wheat, high concentration of ALA reduces the plant growth, whereas application of ALA at low concentrations enhances the growth (Takenouchi et al. 2011). This observation is agreed with previous reports in other plant species (Hotta et al. 1997b; Hara et al. 2010). Ectopic expression of TaALAD1 resulted in increased tolerance to ALA application at high concentrations in transgenic tobacco plants because the introduced TaALAD1 provided the ALAD activity (Takenouchi et al. 2011). Genetic engineering of the tetrapyrrole biosynthetic pathway through modification of both GluTR and ALAD1 might allow us to regulate ALA concentration in plant tissues. If ALA concentration is slightly increased in plants by the genetic engineering, the plant growth can be expectedly improved like the application of ALA at low concentrations.
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