Introduction

The waxy gene encodes the waxy (Wx) protein, or granule bound starch synthase I (GBSSI), which is responsible for amylose biosynthesis in storage tissues. Waxy mutations may affect amylose levels, and waxy mutants with little or no amylose have been described in a number of plant species (Eriksson 1970). The ease of identifying amylose-free mutants using iodine staining has facilitated the use of waxy mutants in genetic studies of diploid plants such as maize, rice and barley (Eriksson 1970). More recently, waxy mutations have been analyzed in detail at the molecular level. In maize, many spontaneous waxy mutations are caused by transposable elements (Fedoroff et al. 1983; Wessler and Varagona 1985; Wessler et al. 1987), whereas a single nucleotide substitution leads to aberrant splicing and reduces both waxy mRNA level and amylose content in rice (Cai et al. 1998; Hirano et al. 1998; Isshiki et al. 1998). In barley, a deletion in the region of the promoter and first intron results in lowered amylose levels, while a single nucleotide substitution in the coding region produces amylose-free cultivars (Patron et al. 2002; Domon et al. 2002).

The development of a method for the separation of the Wx proteins in common or emmer wheat (Nakamura et al. 1993a, 1993b) was followed by the identification of several null alleles for Wx protein (Yamamori et al. 1994, 1995; Demeke et al. 1997; Graybosch et al. 1998; Zhao et al. 1998; Nieto-Taladriz et al. 2000; Urbano et al. 2002). Combining null alleles from each genome allowed the production of waxy mutants in common and tetraploid wheat (Nakamura et al. 1995). A molecular analysis of the three null alleles for the Wx-A1, Wx-B1 and Wx-D1 genes of the hexaploid mutant identified a deletion in the null Wx-A1 gene (Vrinten et al. 1999), and allowed the development of PCR markers that were used to analyze waxy mutations in common wheat germplasm (Nakamura et al. 2002; Saito et al. 2004). In consequence, a new type of mutation induced by an insertion was identified in the Wx-A1 gene of Turkish cultivars (Saito et al. 2004). It appeared that the deletion and the insertion mutations occurred separately and while the deletion spread to several areas of the world, the insertion was conserved only in Turkish cultivars.

Although fewer lines of emmer than common wheat have been analyzed for waxy mutations, several accessions that carry null alleles for Wx-A1 have been identified in the emmer wheat types Triticum dicoccoides Körn., Triticum dicoccum Schübl. and Triticum durum Desf. (Yamamori et al. 1995; Urbano et al. 2002). From an evolutionary point of view, it would be interesting to deduce the relationship of mutations in emmer versus common wheat, or among emmer wheat lines. The Wx-A1 mutation in Turkish durum wheat may be due to an insertion since amplification of a region of the null Wx-A1 allele produced a larger fragment than did the wild-type allele (Urbano et al. 2002). However, besides this PCR data, there are no molecular-based analyses to indicate the origin of the Wx-A1 mutations reported in emmer wheat. In this study, we characterize the Wx-A1 mutations occurring in T. dicoccoides and T. dicoccum and discuss the effects of these lesions on waxy gene expression.

Materials and methods

Plant materials

Seed accessions of T. dicoccoides, T. dicoccum and T. durum were obtained from the Plant Germplasm Institute, Faculty of Agriculture, Kyoto University and the gene bank of the National Institute of Agrobiological Sciences (NIAS) (Tsukuba, Japan). The tetraploid waxy wheat used in this study was selected from the BC5F3 generation of a cross between durum wheat (T. durum Desf. cv. Plenty) with our original waxy tetraploid wheat (Nakamura et al. 1995).

SDS–PAGE

Preparation of starch granules and separation of Wx proteins by low-bis acrylamide SDS-PAGE was performed as described by Nakamura et al. (1993a, 1993b).

DNA extraction and amplification of Wx-A1 gene

Plant DNA was extracted from young leaf tissue using the Nucleon Phytopure Plant DNA Extraction kit (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. The primers and PCR conditions used to amplify null Wx-A1 alleles in hexaploid wheat were the same as those used by Nakamura et al. (2002). To obtain genomic sequence information from emmer wheat, the coding region of the Wx-A1 gene was amplified using KOD-Plus polymerase (Toyobo, Osaka) with the primers 5′-ATGGCGGCTCTGGTCACGTC-3′ and 5′-TCAGGGAGCGGCGACGTTC-3′, which were designed based on the Wx-A1 sequence of common wheat.

Cloning of PCR products and sequencing analysis

PCR products were cloned using the Zero Blunt TOPO PCR Cloning Kit for sequencing or the TOPO TA Cloning Kit for sequencing (Invitrogen, Carlsbad, Calif.). Inserts were sequenced using a BigDye Terminator v1.1 Cycle Sequencing Kit and an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, Calif.).

RNA extraction and RT-PCR

Total RNA was extracted from developing seeds at 10 days post-anthesis (10 DPA) using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA was treated with DNase from a DNA-free kit (Ambion, Austin, Tex.), and 5 μg of total RNA was used for reverse transcription reactions. First-strand cDNA was synthesized at 50°C from random hexamers using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). After the reaction was terminated at 85°C and cooled at room temperature, the remaining RNA was digested with RNase H. The synthesized cDNA was used for a comparative PCR between Wx-A1 and 18S rRNA, which was employed as an endogenous internal standard. The Wx-A1-specific primers 5′-TCCGAGATCAAGGTCGTTGACA-3′ and 5′-CCGGTCTTGCCTTCCACGATA-3′ were used for the detection of Wx-A1 transcript, and the Quantum RNA 18S internal standards primer set (Ambion) was used for 18S rRNA amplification. Each 50 μl reaction contained 1 μl of the RT reaction, 1× KOD Dash buffer, 0.2 mM dNTP, 0.2 μM of each Wx-A1 primer, 0.12 μM 18S primer mix, 0.28 μM 18S competimer and 1 U KOD Dash polymerase (Toyobo). The PCR cycle consisted of an initial 1 min denaturation at 94°C, followed by 25 or 30 cycles of 98°C for 10 s, 62°C for 2 s and 74°C for 30 s. PCR products were separated on a 3% (w/v) agarose gel and quantified with an Agilent 2100 Bioanalyzer using the DNA 7500 LabChip kit (Agilent Technologies, Palo Alto, Calif., USA).

Results

PCR analysis of Wx-A1

Previous studies of emmer wheat germplasm collected throughout the world identified several lines lacking the Wx-A1 protein (Yamamori et al. 1995, 1998; our unpublished data), including a T. dicoccoides accession from Israel (KU13454) and seven T. dicoccum accessions (Table 1), one from Ethiopia (KU9004) and six from Yugoslavia (KU14293, 14294, 14298, 14299, 14302, 14303). None of the accessions analyzed lacked the Wx-B1 protein. Protein from the null Wx-A1 lines was re-analyzed to confirm that these lines lacked the Wx-A1 protein (Fig. 1).

Table 1 Number of null waxy alleles in emmer wheat
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Fig. 1
figure 1

SDS-PAGE analysis of Wx protein bound to starch granules. CS: Chinese Spring, KU13453: Triticum dicoccoides, KU13454: T. dicoccoides, KU14306: T. dicoccum, KU14294: T. dicoccum, waxy tetraploid waxy wheat, Plenty T. durum

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Two different null mutations within the Wx-A1 gene of common wheat have been characterized; one is a 19-bp deletion (Vrinten et al. 1999) which is found in varieties from throughout the world and the second is a 173-bp transposon-like insertion found only in Turkish germplasm (Saito et al. 2004). A primer set capable of distinguishing both types of mutation (Nakamura et al. 2002) was used to analyze DNA from null (KU13454) and wild-type (KU13453) T. dicoccoides lines, and from null (KU14294) and wild-type (KU14306) T. dicoccum lines (Fig. 2). All lines produced a fragment of approximately 389 bp, which is the size expected based on the wild-type hexaploid Wx-A1 genomic sequence. Neither the 19-bp deletion nor the 173-bp insertion were observed, indicating the null Wx-A1 alleles identified in T. dicoccoides and T. dicoccum have a different origin than those found in T. aestivum germplasm.

Fig. 2
figure 2

PCR assay of Wx-A1 genes in emmer wheat accessions. The primer set used here distinguishes common wheat lines carrying null Wx-A1 alleles due to a 19-bp deletion (Norin 18) or a 173-bp insertion (Turkey 124). However, no size differences were detectable between amplification products of wild-type and null Wx-A1 alleles of emmer wheat. Wt (wild-type) T. dicoccoides: KU13453, null T. dicoccoides: KU13454, wt T. dicoccum: KU14306, null T. dicoccum: KU14294

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Sequence analysis of mutant Wx-A1 gene

To obtain sequences of the coding regions of the null Wx-A1 alleles, we designed primers that map to sequences that are conserved among the three T. aestivum waxy genes (see section Materials and methods). A product with the expected size of approximately 2.8 kb was amplified from all four accessions. The amplified fragments were expected to contain waxy sequences derived from both Wx-A1 and Wx-B1 genes. Sequences of several clones from each accession were compared with those of T. aestivum, allowing us to clearly classify clones as Wx-A1 or Wx-B1 genes. The positions of exons in the Wx-A1 genomic sequences were predicted based on T. aestivum waxy cDNA and genomic sequences from publicly available databases. The nucleotide sequence from the wild-type alleles of KU13453 and KU14306 showed several differences from T. aestivum cultivar Chinese Spring (data not shown), but these nucleotide changes resulted in only two amino acid substitutions; positions 60 and 61 are phenylalanine and aspartic acid in T. aestivum but glycine and asparagine in both T. dicoccoides and T. dicoccum (Fig. 3). Amino acid sequences of the two emmer accessions were identical, indicating a high level of conservation of Wx-A1 protein sequences.

Fig. 3
figure 3

Alignment of deduced amino acid sequences of Wx-A1 genes. Identical residues are represented by dots. Positions of premature termination codons and normal stop codons are indicated by outlined asterisks and black asterisks, respectively

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The alignment also revealed several nucleotide differences between the null and wild-type Wx-A1 genes (Fig. 4). In KU13454, one missense substitution, two translationally silent substitutions and a single nucleotide insertion were found (Fig. 4a). The insertion led to a frameshift mutation and a premature termination codon was generated at amino acid position 523 (Figs. 3 and 4b).

Fig. 4
figure 4

Sequence comparison between wild-type and null Wx-A1 alleles of emmer accessions. a Schematic illustration of Wx-A1 gene and locations of nucleotide sequence differences observed in mutated Wx-A1 genes from KU13454 and KU14294. Boxes indicate exons, and gray shading represents coding regions. Insertions and deletions, as well as missense and translationally silent changes, are indicated above (KU13454) or below (KU14294) the exon boxes. b Frameshift mutation in the null Wx-A1 allele of KU13454. A 1-bp insertion in exon 10 changes the reading frame, and creates a premature termination codon at the 3′ end of exon 10. c Frameshift mutation in the null Wx-A1 allele of KU14294. A 1-bp deletion at nucleotide 23 of exon 4 creates a premature termination codon

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In KU14294, a single nucleotide deletion at position 23 of exon 4 also caused a frameshift mutation (Fig. 4a, c). A premature termination codon was generated at amino acid position 144 (Figs. 3, 4c). Although mRNA is transcribed from both genes, it is unlikely that functional Wx protein would be produced from either transcript.

Expression of Wx-A1 gene in emmer wheat

In waxy common wheat, the amount of waxy transcript is greatly reduced in comparison to non-waxy wheat (Vrinten et al. 1999). However, the amylose-free barley cultivar Yon M Kei, which also carries a premature termination codon in its waxy gene, shows the same level of waxy transcript as wild-type barley (Patron et al. 2002). Therefore, relative RT-PCR experiments were performed to determine if Wx-A1 transcript levels were affected by the mutations described here. The coding region of the Wx-A1 gene was amplified from 10 DPA endosperm total RNA using Wx-A1 specific primers, and 18S rRNA was co-amplified as an internal standard. The amount of 18S rRNA was almost identical in all RNA samples examined. However, critical differences were observed in the amount of Wx-A1 transcript found in wild-type versus mutant plants (Fig. 5a); gel electrophoresis followed by ethidium bromide staining detected a small amount of amplification product in KU13454 reactions, while no product was visible in KU14294 samples. However, product could be detected even in KU14294 when samples were analyzed using an Agilent Bioanalyzer. The ratio of Wx-A1 to 18S rRNA molarity was taken as the relative amount of Wx-A1 transcript (Fig. 6). From this, the levels of Wx-A1 transcripts in KU13454 and KU14294 were calculated as approximately 6.5% of KU 13453 and 1.4% of KU14306, respectively.

Fig. 5
figure 5

Expression of Wx-A1 genes in KU13454 and KU14294. The amounts of Wx-A1 transcripts and 18S rRNA were analyzed by multiplex RT-PCR. PCR was performed with 25 (a) or 30 reaction cycles (b). The arrowhead indicates PCR product amplified from Wx-A1 transcripts with retained introns

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Fig. 6
figure 6

Quantitative comparison of mature Wx-A1 transcripts. Comparative RT-PCR (25 PCR cycles) was conducted with Wx-A1 and 18S rRNA-specific primer sets and amplified products were analyzed using an Agilent 2100 Bioanalyzer. The amounts of Wx-A1 and 18S rRNA PCR products were calculated in molarity (nmol/l) and the Wx-A1 product amount was normalized to the rRNA product amount

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When the number of PCR cycles was increased to 30, an amplification product with a higher molecular weight than the 1,145-bp fully spliced Wx-A1 transcript was detected in KU14294 (Fig. 5b, arrowhead). Sequencing revealed this product consisted of at least three types of sequence carrying one or two unspliced introns (Fig. 7). A similar-sized product was also found in KU13454 when the number of cycles and the amount of product loaded were increased (Fig. 7). This fragment included two different amplification products containing intron sequences as well as a product that resulted from the use of a variant splicing site.

Fig. 7
figure 7

Schematic representation of Wx-A1 fragments amplified from total RNA of KU13454 and KU14294 using RT-PCR. Wx-A1 specific primers, indicated by horizontal arrows, were used. Boxes indicate exons, and vertical lines indicate the positions of premature termination codons. One or two introns were retained in transcripts (lines), and the outlined arrow indicates an intron that originated from an aberrant splicing site

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Discussion

The wild emmer wheat T. dicoccoides is thought to have primarily originated in the western arc of the Fertile Crescent from the upper Jordan Valley to southeastern Anatolia (Johnson 1975). T. dicoccoides is the progenitor of the domesticated emmer wheat types T. dicoccum and T. durum. Archaeological evidence indicates that emmer wheat has been cultivated since the early Neolithic era, and was the most important crop until the early Bronze Age in the Near East (Bell 1987). Although tetraploid wheat evolution has been studied using waxy gene sequences (Yan and Bhave 2000, 2001), there are few reports relating to waxy mutations in tetraploid wheat. In this report, we were able to elucidate the causes of the mutations of the Wx-A1 genes in T. dicoccoides and T. dicoccum. In both cases, the mutation originated from a frameshift mutation caused by a single nucleotide insertion or deletion (Fig. 4). However, the Wx-A1 mutation in T. dicoccum was not related to that in its progenitor, T. dicoccoides, and the two mutations clearly arose independently.

The frameshift mutations described here generated premature termination codons in the RNA sequences (Fig. 4), which would reduce the length of the translation products by 82 and 461 amino acids in KU13454 and KU14294, respectively (Fig. 3). Both mutated genes were transcribed, but dramatic reductions in transcript levels were observed, suggesting that mRNA was recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). NMD or RNA surveillance is a transcript quality control system (reviewed in Wilusz et al. 2001; Schell et al. 2002; Wagner and Lykke-Andersen 2002; Maquat 2004) that prevents the accumulation of high levels of truncated proteins translated from mRNAs containing a premature termination codon. Such truncated proteins may exert a dominant-negative effect (Maquat 2004), and NMD prevents their accumulation by selectively eliminating aberrant transcripts. In mammals, the position of the premature termination codon and the presence of introns are important in eliciting NMD. Only those premature termination codons positioned more than 50–55 nucleotides upstream of the final exon-exon junction result in NMD, and premature termination codons occurring in the last exon or in intronless genes cannot elicit NMD (Maquat 2004).

The mechanisms involved in NMD in plants are still under investigation. Evidence that premature termination codons can reduce mRNA level was initially presented in studies of the Kunitz trypsin inhibitor (KTi) gene in soybean (Jofuku et al. 1989), the phytohemagglutinin (PHA) gene in common bean (Voelker et al. 1990), and the ferredoxin-1 (Fed-1) gene in Pisum (Dickey et al. 1994). These are all intronless genes, therefore the rules governing NMD in plants may differ from those in mammals. Reduction in mRNA abundance also occurs in intron-containing genes such as the phytochrome A (Dehesh et al. 1993) and AUX1 (Marchant and Bennett 1998) genes of Arabidopsis, and the waxy gene of rice (Isshiki et al. 2001). In these cases, the premature termination codons are located more than 50–55 nucleotides upstream of the final exon-exon junction, consistent with the mammalian NMD rules. The premature termination codons identified in the mutated Wx-A1 genes of KU13454 and KU14294 also appear to fit these conditions, and drastic reductions in transcript levels were observed in both cases. Therefore, the deficiency of Wx-A1 protein observed here is likely to be largely due to rapid mRNA degradation because of NMD. The amount of transcript detected was very small, but if some translation still occurred, the resulting protein would not likely be functional since it would lack the C-terminal region. The active form of GBSSI is tightly bound to the starch matrix, and the C-terminal region is thought to be involved in this attachment (van de Wal et al. 1998).

Wx-A1 fragments retaining introns were amplified from KU13454 and KU14294 but not from the wild-type alleles (Figs. 5b, 7). In addition, KU13454 carried a 1,261-bp fragment (Fig. 7) in which a different splicing site replaced the primary site between exons seven and eight (Fig. 7). Point mutations in the coding region may affect not only amino acid sequence but also pre-mRNA splicing (Cartegni et al. 2002; Faustino and Cooper 2003). In particular, frameshift and nonsense mutations can elicit nonsense-associated altered splicing (NAS) in mammalian cells, including exon skipping, alternative splicing site choice and intron retention (Cartegni et al. 2002; Maquat 2002). NAS may be involved in the production of the aberrantly spliced Wx-A1 transcripts in KU13454 and KU14294, although we cannot rule out the possibility that the relatively low levels of correctly processed transcripts in these genotypes led to selective amplification of incompletely or improperly processed transcripts.

Although Wx-A1 mutations were identified in eight accessions of emmer wheat (Table 1), only two mutants were subjected to molecular analysis in this study. The six T. dicoccum accessions from Yugoslavia were all collected from the same region, and it is likely that they all carry the same point mutation. Unfortunately, due to a limited number of seed and a lack of seed germination, we were unable to analyze the T. dicoccum accession from Ethiopia, and the molecular details of the mutation carried by this line remain unknown. The cause of a Wx-A1 mutation carried by a durum wheat line (Urbano et al. 2002) is also unknown. Further analysis of these mutants will reveal whether the lesions they carry are related to the mutations described here.