Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat

Gene 234 (1999) 71–79 www.elsevier.com/locate/gene

Isolation and characterization of the three Waxy genes encoding the granule-bound starch synthase in hexaploid wheat k J. Murai, T. Taira, D. Ohta * Laboratory of Plant Genes and Physiology, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Received 28 January 1999; received in revised form 12 April 1999; accepted 3 May 1999; Received by W. Martin

Abstract Complete genomic DNA sequences of three homoeologous Waxy structural genes, located on the chromosomes 7A, 4A, and 7D in hexaploid wheat (Triticum aestivum L. cv. Chinese Spring), were separately determined and analyzed. Those structural genes in lengths from start to stop codon were 2781 bp in Wx-7A, 2794 bp in Wx-4A, and 2862 bp in Wx-7D, each of which consisted of 11 exons and ten introns. They were closely similar to one another in the nucleotide sequences, with 95.6–96.3% homology in mature protein regions, 88.7–93.0% in transit-peptide regions, and 70.5–75.2% in the introns. These wheat Waxy genes were GC-rich when compared with standard values for plant genomes reported so far. This was reflected in the extremely high G/C occupation frequency at the third position of the codons in the coding regions. The sequence divergence in the exon regions was mostly due to the substitution of nucleotides, whereas that found in the introns was attributed to substitution, insertion and/or deletion of nucleotides. Only the Wx-4A gene contained a trinucleotide insertion (CAA) in the region encoding the transit peptide. Most of the substitutions observed in the exon regions were categorized as synonymous, and higher sequence similarities (96.5–97.4%) were conserved at the protein level. The phylogenetic tree obtained in terms of the amino acid sequence variations showed a well-resolved phylogenetic relationship among wheat Waxy genes and those from other plants. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amylose synthase; GC content; Gene structure; Genomic nucleotide sequence; Triticum aestivum

1. Introduction Genome constitutions of polyploid wheats are AABB in tetraploid (macaroni wheat) and AABBDD in hexaploid (bread wheat). It has been argued that tetraploid wheat evolved through crosses between diploid progenitors, one bearing the genome A and the other bearing the genome S (the genome in the Sitopsis section of the genus Aegilops), and hexaploid wheat then emerged upon the addition of the D genome derived from Ae. Abbreviations: IPCR, inverse PCR; N7A/T7B, nullisomic 7A-tetrasomic 7B; N7D/T7B, nullisomic 7D-tetrasomic 7B; PCR, polymerase chain reaction; 3∞-RACE, 3∞-rapid amplification of cDNA ends; RT, reverse transcriptase; UPGMA, unweighted pair group method with arithmetic mean. k The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank Nucleotide Sequence Databases with the following Accession Nos: Wx-7A, AB019622; Wx-4A, AB019623; Wx-7D, AB019624. * Corresponding author. Tel.: +81-722-54-9409; fax: +81-722-54-9409. E-mail address: [email protected] (D. Ohta)

squarrosa to a tetraploid wheat ( Kimber, 1974). Each genome of hexaploid wheat possesses one Waxy locus, encoding Waxy protein, in principle, and three Waxy genes, viz., Wx-7A, Wx-4A, and Wx-7D (tentatively named in this paper), are located on the chromosomes 7A, 4A (translocated from 7B), and 7D, respectively, as evidenced by the restriction fragment length polymorphism analyses of homoeologous group 7 chromosomes of hexaploid wheat (Chao et al., 1989). Granule-bound starch synthase (GBSSI, EC 2.4.1.21), also called Waxy protein ( Echt and Schwartz, 1981), is a nuclear-encoded enzyme of about 60 kDa, which plays a crucial role in the amylose synthesis in the plastids of plants ( Vos-Scheperkeuter et al., 1986). The cDNA and genomic DNA sequences encoding the Waxy proteins of maize ( Klo¨sgen et al., 1986), barley ( Rohde et al., 1988), rice ( Wang et al., 1990), sorghum ( Hsieh et al., 1996), potato (van der Leij et al., 1991), pea (Dry et al., 1992), and cassava (Salehuzzaman et al., 1993) have been reported. A Waxy cDNA from hexaploid wheat was also cloned and sequenced (Clark et al.,

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 17 8 - X

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2. Materials and methods

was performed in a reaction mixture (50 ml ) containing 50 ng of genomic DNA as the template, 0.2 mM of dNTP, 0.5 mM of each primer, 1.25 unit of Ampli-Taq DNA polymerase (Perkin Elmer, Foster, CA), 50 mM KCl, 1.5 mM MgCl , and 10 mM Tris–HCl (pH 8.3). 2 Each reaction was overlaid with 50 ml of light mineral oil (Nacalai Tesque, Kyoto, Japan) to prevent evaporation. An initial denaturation period of 1 min at 94°C was followed by 43 cycles of 1 min at 64°C, and a final elongation step was 1 min at 72°C. PCR fragments were extracted from agarose gel slices with a QiaexII Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned using the pT7blue T-vector system (Novagen, Madison, WI ) and an E. coli strain, DH5a according to the manufacturer’s protocol.

2.1. Plant materials

2.4. Cloning the 5∞- and 3∞ non-coding regions

Hexaploid wheat (Triticum aestivum cv. Chinese Spring) and its nullisomic-tetrasomic lines, nullisomic 7A-tetrasomic 7B (N7A/T7B) and nullisomic 7Dtetrasomic 7B (N7D/T7B), were used. Plants were grown in a greenhouse during the winter season. Immature seeds of 10 days post-anthesis were harvested and stored at −85°C until use. Etiolated seedlings (8 cm) were used for isolation of genomic DNA.

We employed inverse PCR (IPCR) and 3∞ rapid amplification of cDNA ends (3∞-RACE) to amplify 5∞and 3∞ non-coding regions, respectively. In the IPCR, HindIII digested-genomic DNA samples were circularized using a Ligation Kit ver. 2 ( Takara Shuzo, Kyoto, Japan) and used as the template for the PCR. When genomic DNA was used as the template, primers were newly designed referring to the DNA sequences determined by the first round IPCR. Reverse transcriptase (RT )-PCR and 3∞-RACE were performed using total RNA as the template and an RNA PCR Kit (AMV ) ver. 2.1 ( Takara Shuzo) following the manufacturer’s protocol. The amplified DNA fragments were subcloned using the pT7blue T-vector system as described above. Products from IPCR and RT-PCR were completely sequenced.

1991; Ainsworth et al., 1993), but it has not been clarified which Waxy gene(s) of the hexaploid genome encoded this cloned cDNA. In this paper, we report the genomic nucleotide sequences of three Waxy genes (Wx-7A, Wx-4A, and Wx-7D) from hexaploid wheat. Wx-7A, Wx-4A, and Wx-7D genes are considered as homoeologous genes, which are inferred to have evolved from diploid wheat, including a closely related species. We also present genomic organizations of these Waxy genes and characterize the structures comparing with those reported with other plant species.

2.2. Preparation of genomic DNA and RNA Genomic DNA was extracted from leaf blades of etiolated seedlings with an extraction buffer containing 2% cetyltrimethylammonium bromide, 0.1 M Tris–HCl (pH 8.0), 1.4 M NaCl, 1% polyvinylpyrrolidone, 0.2% 2-mercaptoethanol. DNA was further purified by phenol/chloroform extraction and ethanol precipitation and resuspended in 10 mM Tris–HCl (pH 8.0) containing 1 mM EDTA (Rogers and Bendich, 1985). Total RNA was isolated from immature seeds with a nucleic acid extraction mixture (Isogen, Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. 2.3. PCR strategy Assuming a structural conservation of the Waxy genes between hexaploid wheat and barley, we set PCR primer positions by comparing a Waxy cDNA sequence of hexaploid wheat (Clark et al., 1991) and a genomic Waxy DNA sequence of barley (Rohde et al., 1988). For each PCR reaction, a primer set was thus designed to cover at least two putative intron regions. In our PCR strategy, two adjacent PCR fragments were designed to contain at least one intron in common. Since intron regions are usually less homologous than exon regions, we were able to distinguish the original Waxy genes by comparing the sequence divergence in the intron regions in the amplified PCR fragments. PCR

2.5. Sequencing of genomic DNA and cDNA Primers were labelled with a 5∞-oligonucleotide Texas Red Labelling Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The sequencing was performed with a Thermo Sequenase Core Sequencing Kit with 7-deazadGTP (Amersham Pharmacia Biotech) according to the manufacturer’s protocol and a SQ5500 automated sequencer (Hitachi, Tokyo, Japan). 2.6. Construction of phylogenetic tree A phylogenetic tree among Waxy genes was obtained by analyzing the amino acid sequence divergence of the Waxy proteins, and a dendrogram was constructed using the unweighted pair group method with arithmetic mean ( UPGMA) (Sneath and Sokal, 1973) incorporated into the GENETYX-MAC program ver. 8 (Software Development, Tokyo, Japan). Genetic distances between pairs of sequences were calculated according to Kimura’s formula ( Kimura, 1983).

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3. Results and discussion

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mined to be 2781 bp for Wx-7A, 2794 bp for Wx-4A, and 2862 bp for Wx-7D in lengths.

3.1. Nucleotide sequences of three homoeologous Waxy genes

3.2. Analysis of three Waxy structural genes

We have determined the complete nucleotide sequences of the three homoeologous Waxy structural genes (Wx-7A, Wx-4A, and Wx-7D) of hexaploid wheat. In the PCR-cloning strategy, we assumed that the overall genomic organizations of the Waxy genes were conserved between hexaploid wheat and barley. Thus, PCR primers ( Fig. 1) were designed by comparing a Waxy cDNA sequence of hexaploid wheat (Clark et al., 1991) and a genomic Waxy DNA sequence of barley (Rohde et al., 1988). With this PCR strategy, we should be able to amplify 18 different regions covering a whole structural gene. Each of these 18 independent fragments contained overlapped regions including putative two different introns as shown in Fig. 1. PCR amplification was carried out using genomic DNA from either N7D/T7B line or N7A/T7B line as the template. Five to ten DNA fragments were independently cloned from every PCR reaction, and the insert DNA sequences were completely determined. Whole structures of the Waxy genes were constructed by connecting the sequences of the PCR fragments with the aid of an identity of the overlapped intron regions as designed to be present in each PCR product (Fig. 1). The Waxy genes were distinguished by comparing the sequence divergence in the intron regions in the amplified PCR fragments. The N7D/T7B line gave the genomic sequences of both Wx-7A and Wx-4A, and the N7A/T7B line gave those of both Wx-4A and Wx-7D. No other sequences appeared ( Fig. 2) in our experiments. The structural genes including both exons and introns were thus deter-

Intron positions were identified by comparing the genomic sequences ( Fig. 2) with the Waxy cDNA sequence of hexaploid wheat (Clark et al., 1991). Total intron numbers in the Waxy genes from hexaploid wheat were determined to be ten divided by 11 exons (Fig. 2). Dinucleotide sequences found at the 5∞- and 3∞-ends of nine out of ten introns followed the universal GT–AG rule (Breathnach and Chambon, 1981). However, the GT–AG rule did not fit with the sequence for the 3∞-dinucleotide end of the putative fourth intron ( Fig. 3a). Thus, the Waxy genes determined from N7D/T7B line, possessing Wx-7A and Wx-4A genes, were proved to carry TG at the 3∞-end of the fourth intron. This was also the case for the common Waxy gene present in both N7D/T7B line and N7A/T7B line, possessing Wx-4A and Wx-7D genes. However, the sequence corresponding to the same site specifically detected from N7A/T7B line was GG when compared with the reported Waxy cDNA sequence (Clark et al., 1991) (Fig. 3a). These results indicated that the three Waxy genes contained the unusual dinucleotide sequences at the 3∞-end of the fourth intron. In order to investigate these unusual dinucleotide sequences, we set primers on the exon regions located both upstream (Primers A and B) and downstream (Primer C ) of the fourth intron region (Fig. 3b) and performed RT-PCR using the mRNA extracted from immature seeds from N7D/T7B line to confirm them with the Waxy cDNA sequences ( Fig. 3c). From this experiment, we found that the splicing site was located

Fig. 1. Primer positions (triangles) and cloning sites (indicated by dotted lines) on Waxy gene from hexaploid wheat. Thirty-four different primers were used for the PCR to determine the Waxy gene structures. Primer 34 (5∞-GTTTTCCCAGTCACGAC-3∞) annealed with the oligo dT-adaptor primer sequence (Takara Shuzo) and was used for the PCR using the 3∞-RACE reaction products amplified with the M13 M4 primer as the template. Rectangulars indicate exon regions. The trinucleotide, GCC, indicates the codon of the N-terminus of a mature Waxy protein predicted from the sequence reported (Fujita et al., 1996). Bars indicate cloned DNA fragments by PCR.

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Fig. 2. Alignment of genomic DNA sequences of three homoeologous Waxy genes (Wx-7A, Wx-4A, and Wx-7D) from hexaploid wheat. Gaps were introduced to maximize the agreement of the sequence alignment. The exon and intron regions are shown by capital letters boxed with gray color and small letters, respectively. Identical bases are presented by dots.

at further 33 bp downstream than the reported position for the Waxy cDNA sequence (Clark et al., 1991). Thus, no amplified DNA fragments appeared ( Fig. 3c, lane 2b) when RT-PCR was carried out using Primer C together with Primer B, which was set inside the 33-bp region (Fig. 3b). However, a clear DNA band (Fig. 3c, lane 2a) appeared when the same primer set was used with the genomic DNA fragment specifically amplified

from N7D/T7B line as the template, which had been isolated for the determination of the gene structures ( Figs. 1 and 2). These results indicated that the 33-bp region was not present in the mature mRNA for the Waxy genes. We, therefore, concluded that the 33-bp region reported as an exon was a part of the fourth intron and that the splicing site of the 3∞-end of the fourth intron of Wx-7A gene obeyed the universal AG

J. Murai et al. / Gene 234 (1999) 71–79

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Fig. 2. (continued )

instead of TG. The 33-bp sequence was found to be the intron in Wx-4A and Wx-7D genes as well in similar experiments (data not shown). Next, we compared the nucleotide sequence of the fifth exon of Wx-7A gene with the same exon region of the reported Waxy cDNA (Clark et al., 1991) from which the 33-bp region had been eliminated. Both sequences were completely identical, except two bases, and the reported Waxy cDNA (Clark et al., 1991) was identified as the product of Wx-7A gene. Furthermore, the 33-bp long sequence is not found in the corresponding exon regions of any of the Waxy genes from other plant species ( Klo¨sgen et al., 1986; Rohde et al., 1988; Wang et al., 1990; van der Leij et al., 1991; Dry et al., 1992; Hsieh et al., 1996; Salehuzzaman et al., 1993). It has been established that Waxy protein is a nuclearencoded enzyme synthesized in cytoplasm as a precursor protein with the N-terminal transit peptide ( Klo¨sgen et al., 1986). Fujita et al. (1996) reported that three Waxy gene products could be distinguished from one another by partial amino acid sequencing of the mature Waxy proteins from nullisomic-tetrasomic lines of hexaploid wheat (Fujita et al., 1996). Specifically, they proposed two indicator residues: the fifth residue at the N-terminus and the 185th residue from the C-terminus were glycine and valine for Wx-7A protein, alanine and leucine for Wx-4A protein, and glycine and leucine for Wx-7D protein, respectively. We compared the amino acid sequences deduced from the Waxy genes of hexa-

ploid wheat with the partial amino acid sequences of the mature Waxy proteins ( Fujita et al., 1996). The amino acid residues at the two indicator sites in the deduced amino acid sequence completely agreed with the sequences of those Waxy proteins (Fig. 4). 3.3. Characteristics of Wx-7A, Wx-4A and Wx-7D genes As mentioned above, the Waxy structural genes encode precursor forms composed of the regions corresponding to transit peptides and mature forms. We compared the deduced amino acid sequences encoded by Wx-7A, Wx-4A and Wx-7D genes with the N-terminal sequences of the mature Waxy proteins of hexaploid bread wheat ( Fujita et al., 1996) and found that the regions encoding the transit peptides and the mature proteins were 210 and 1605 bp in Wx-7A, 213 and 1605 bp in Wx-4A, and 210 and 1605 bp in Wx-7D, respectively. The sequences encoding the transit peptides and the mature proteins were highly homologous among those Waxy genes, i.e. 88.7 and 95.9% between Wx-7A and Wx-4A, 91.0 and 95.6% between Wx-7A and Wx-7D, and 93.0 and 96.3% between Wx-4A and Wx-7D, respectively ( Table 1). In particular, the regions coding for the mature proteins showed a higher homology compared with the transit peptide sequences. The sequence divergence in the exon regions between these three Waxy genes was mainly attributed to the substitu-

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Fig. 3. (a) Regions containing the dinucleotides shown by gray color disagreed with the GT–AG rule (Breathnach and Chambon, 1981). Small and large letters indicate the fourth intron and the fifth exon from the start codon, respectively; the fifth exon previously reported (Clark et al., 1991) was shown. (b) Positions of Primer A (5∞-GCAGCACTTGAGGTGCCCAGGAT-3∞), Primer B (5∞-CCATGCTATGCCGTGCCGTGC-3∞), and Primer C (5∞-CTTGGCCGTCCTATAGATGCCAT-3∞) were designed from the Waxy gene structure. The gray region was examined to determine whether it should be ascribed to a part of the fourth intron or the fifth exon regions. (c) PCR amplification using genomic DNA ( lanes 1a and 2a) and cDNA ( lanes 1b and 2b) from N7D/T7B as the templates. The primers A and C were used in lanes 1a and 1b, and the primers B and C in lanes 2a and 2b. A wX174/HincII digest was used as a molecular size marker.

Fig. 4. Alignment of deduced amino acid sequences of three homoeologous Waxy genes, Wx-7A, Wx-4A, and Wx-7D, in hexaploid wheat. Amino acids are shown as one-letter codes. Identical residues are presented by dots.

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tion of nucleotide, whereas that in the intron regions was due to substitution, insertion and/or deletion of nucleotides. The insertion of trinucleotide (CAA) in the transit peptide region was only found in the Wx-4A gene (Fig. 2). Since the progenitor species of B-genome donor remains controversial, the occurrence of the trinucleotide insertion could serve as a useful marker for searching the progenitor. Ratios of non-synonymous to synonymous substitution in the transit peptide and the mature protein coding regions were 0.663 and 0.134 between Wx-7A and Wx-4A, 0.564 and 0.095 between Wx-7A and Wx-7D, and 0.180 and 0.091 between Wx-4A and Wx-7D, respectively ( Table 1), indicating that the nucleotide substitution rates were higher in the transit peptide regions than in the mature proteins. Homologies in the introns were 75.2% between Wx-7A and Wx-4A, 70.5% between Wx-7A and Wx-7D, and 74.0% between Wx-4A and Wx-7D ( Table 1), suggesting that evolution rates in the introns were greater than those in the exons. In other words, a higher selective pressure was present to have kept the catalytic activity of the Waxy proteins, and the nucleotide sequences were then highly conserved in the exons. The GC contents in the coding regions for the transit peptides and the mature proteins were 68.6 and 63.6% in Wx-7A, 70.4 and 64.7% in Wx-4A, and 71.9 and 63.9% in Wx-7D, respectively. These GC contents exceeded by far the average value (55.3%) calculated with 279 wheat genes deposited in the GenBank database using the Codon Usage Tabulated from GenBank (Nakamura et al., 1998). Intron regions were low in G and C: 48.0% in Wx-7A, 49.5% in Wx-4A, and 45.8% in Wx-7D. The GC contents of the Waxy genes of monocotyledonous plants including wheat, barley, sorghum, maize, and rice were higher than those of dicotyledonous plants such as cassava, potato, and pea ( Table 2). It has been reported that nuclear genes of monocot plants could be grouped into two types: GC-biased genes with higher GC occupation rates at the third codon position and GC-nonbiased genes with well-

balanced GC distribution patterns (Brinkmann et al., 1987; Campbell and Gowri, 1990). The wheat Waxy genes exhibited remarkable bias towards GC: the GC occupation at third-codon positions ranged from 81.4 to 84.3% in the regions of transit peptide and from 91.8 to 94.4% in the mature protein regions ( Table 2). These values were considerably higher than the average value for the 279 wheat genes (62.1%). No GC bias was found with the dicot Waxy genes shown in Table 2. These observations were consistent with the general characteristics of different genes of monocot and dicot plant species (Brinkmann et al., 1987; Campbell and Gowri, 1990; Gardiner-Garden and Frommer, 1992; Jansson et al., 1994). 3.4. Waxy proteins The Waxy genes ( Fig. 2) were predicted to encode Waxy precursor forms consisted of 604 amino acid residues in Wx-7A and Wx-7D and 605 amino acid residues in Wx-4A. The transit peptide sequences were composed of 70 amino acid residues in both Wx-7A and Wx-7D proteins but 71 in Wx-4A protein. The calculated molecular masses for the transit peptides and mature proteins were 7.62 and 58.95 kDa in Wx-7A, 7.57 and 58.8 kDa in Wx-4A, and 7.38 and 58.90 kDa in Wx-7D, respectively. While these calculated molecular mass of the mature proteins were slightly smaller than those estimated by SDS-PAGE (Fujita et al., 1996), the molecular size among three proteins followed the same order irrespective of these two different methods for molecular size estimation. The regions of the transit peptides and the mature proteins were 84.5 and 96.5% similar between Wx-7A and Wx-4A proteins, 87.1 and 96.8% between Wx-7A and Wx-7D proteins, and 94.4 and 97.4% between Wx-4A and Wx-7D proteins, respectively ( Table 1). Then, we compared the primary structures of the mature proteins from hexaploid wheat to those of the Waxy proteins from other plant species, such as barley (Rohde et al., 1988), rice ( Wang et al., 1990), maize ( Klo¨sgen et al., 1986), sorghum (Hsieh et al., 1996), cassava

Table 1 Homologies, similarities, and ratios of non-synonymous to synonymous substitutions between three Waxy genes in hexaploid wheat

Homology of genomic DNA sequence (%)

Wx-7A/4A

Wx-7A/7D

Wx-7A/7D

87.8

85.8

87.6

Intron region Transit peptide

88.7 95.9 75.2 84.5

91.0 95.6 70.5 87.1

93.0 96.3 74.0 94.4

Mature waxy protein Transit peptide Mature waxy protein

96.5 0.663 0.134

96.8 0.564 0.095

97.4 0.180 0.091

From start codon to stop codon Exon region

Similarity of amino acid sequence (%) Non-synonymous/synonymous

Transit peptide coding region Mature waxy protein coding region

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Table 2 GC content (%) in the coding region and at the first, second, and third codon positions Plant

Monocotyledon

Dicotyledon

Coding region

First position

Second position

Third position

ta

m

t

m

t

m

t

m

Wheat (Wx-7A) (Wx-4A) (Wx-7D) Barleyb Maizec Sorghumd Ricee

68.6 70.4 71.9 70.0 80.1 76.6 74.0

63.6 64.7 63.9 63.4 64.4 62.0 59.9

64.3 64.8 65.7 65.7 73.6 72.7 59.7

60.4 60.4 60.2 59.2 60.1 58.8 58.0

60.0 64.8 65.7 64.3 69.4 70.1 64.9

38.5 39.3 38.5 38.6 40.8 40.0 40.5

81.4 81.7 84.3 80.0 97.2 87.0 97.4

91.8 94.4 93.1 92.3 92.1 87.2 81.1

Cassavaf Potatog Peah

44.0 44.6 44.9

46.0 44.8 43.0

39.7 39.0 40.0

55.0 55.0 53.3

48.7 53.2 50.7

39.0 38.2 37.8

43.6 41.6 44.0

43.9 41.2 37.8

a t, transit peptide; m, mature protein. b Rohde et al. (1988). c Klo¨sgen et al. (1986). d Hsieh et al. (1996). e Wang et al. (1990). f Salehuzzaman et al. (1993). g van der Leij et al. (1991). h Dry et al. (1992).

(Salehuzzaman et al., 1993), potato (van der Leij et al., 1991), and pea (Dry et al., 1992), and it was found that the mature protein regions were more than 85% similar among monocot plants. In particular, the wheat Waxy proteins were more than 95% similar to that of barley. However, the similarity between monocot and dicot Waxy mature proteins was less than 71%. It was also shown that the structural conservation in the transit peptide regions was generally low when compared with that observed in the mature protein regions. Such a loose sequence conservation could be sufficient to keep the general properties as a transit peptide (von Heijne and Nishikawa, 1991) during evolution. A phylogenetic tree was constructed on the basis of

the sequence similarity of the mature proteins (Fig. 5), and it was suggested that the Waxy proteins were divided into two groups. Thus, one of the two groups consisted of monocotyledonous plants. This group was further divided into two clusters: one cluster contained maize, sorghum, and rice, and another cluster was composed of species including wheat and barley. The dicot proteins were grouped into a separate cluster. To our knowledge, this is the first report of the complete genomic structures of three Waxy genes of hexaploid wheat. Each genome of hexaploid wheat possesses one Waxy locus, encoding Waxy protein, and three Waxy genes, Wx-7A, Wx-4A, and Wx-7D, are located on the chromosomes 7A, 4A (translocated from 7B), and 7D, respectively. It has been documented that the amount of Waxy protein is a major factor in determining the rate of amylose synthesis and the amylose content of starch in storage organs (Smith et al., 1997). Future studies should focus on the mechanism by which these Waxy genes are regulated in hexaploid wheat, and it is of particular interest as to how these individual isoforms are involved in the starch synthesis in different organs.

Acknowledgements Fig. 5. Phylogenetic tree of the Waxy mature proteins. The amino acid sequences of the mature proteins (Fig. 4) were analyzed using the UPGMA method (Sneath and Sokal, 1973). Numbers indicate genetic distance.

The authors are grateful to Drs Tsuneyuki Yamazaki and Hidenori Tachida for helpful discussion. This work was supported by a Grant-in-Aid for Scientific Research

J. Murai et al. / Gene 234 (1999) 71–79

from the Ministry of Education, Science and Culture of Japan.

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