JXB Advance Access originally published online on January 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 396, pp. 365-375, February 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Isolation and location of three homoeologous dihydroflavonol-4-reductase (DFR) genes of wheat and their tissue-dependent expression
Received 22 July 2003; Accepted 19 October 2003
Research Institute of Bioresources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama, 710-0046, Japan
* To whom correspondence should be addressed. Fax: +81 86 434 1249. E-mail: knoda{at}rib.okayama-u.ac.jp
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Abstract |
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DFR is involved in an important step in the flavonoid biosynthesis pathway upstream of anthocyanin, proanthocyanidin, and phlobaphene production, which contributes to the pigmentation of various plant tissues. Full genomic sequences of three DFRs were isolated in hexaploid wheat. Loci of TaDFRs were found in a more proximal region of the long arm of chromosomes of homoeologous group 3 than the R gene for red grain colour of wheat. These DFRs were designated TaDFR-A, TaDFR-B, and TaDFR-D on chromosome 3A, 3B, and 3D, respectively. In the 5' upstream region of DFR genes, two or three combinations of a G box core element and a putative binding site for a Myb-type transcription factor, P, of maize were found. Expression of DFR reached a maximal level in red grain of wheat cv. Chinese Spring (CS) at 5 d post-anthesis (DPA) and decreased gradually in the grain coat tissue from 10 to 20 DPA, in contrast to a very low expression level of DFR in white wheat grain during the same period. These DFRs differed in their expression. TaDFR-B and -D were expressed predominantly in grains. In developing leaves, DFR expression was light-responsive, and TaDFR-B was more up-regulated in leaves and roots than the other two.
Key words: DFR gene, flavonoid, grain colour, wheat.
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Introduction |
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The red grain colour of wheat has been shown to affect two economically important characteristics of wheat. It directly alters levels of grain dormancy and resistance to preharvest sprouting (Flintham, 2000; Warner et al., 2000; Himi et al., 2002). Grain colour also reduces the brightness of wheat flour due to contamination by the seed coat tissue during the milling process. The red pigment of wheat grain coats has been reported to be a polyphenolic compound, ‘phlobaphene’ or ‘proanthocyanidin’ (Miyamoto and Everson, 1958). In maize, phlobaphene accumulates specifically in the floral organs such as the kernel pericarp and cob tissues. It is synthesized in the early steps of flavonoid synthesis by three enzymes: chalcone synthase (CHS), chalcone flavanone isomerase (CHI), and dihydroflavonol-4-reductase (DFR) (Holton and Cornish, 1995; Chopra et al., 1996; Mol et al., 1998). These findings in maize suggest that the pigment of wheat grain colour is also synthesized by these enzymes in the flavonoid biosynthesis pathway.
Three dominant alleles, R-A1b, R-B1b, and R-D1b of the R genes on the long arms of wheat chromosomes 3A, 3B, and 3D have been verified cytogenetically as being able to confer the red colour to wheat grain (McIntosh et al., 1998). However, it is still not clear whether the R gene is a structural gene for an enzyme or a regulatory factor in flavonoid biosynthesis. Regarding grain colour development, the focus has been on the Vp1 gene of maize as a candidate of the wheat R gene. Vp1 regulates the expression of a Myb-type C1 regulatory gene, which activates the genes in the flavonoid biosynthesis pathway (McCarty et al., 1989). Furthermore, embryo sensitivity to abscisic acid (ABA), grain dormancy, grain maturation, and the expression of the 
-amylase gene are partly under the control of Vp1 (McCarty, 1995). Bailey et al. (1999a) isolated wheat Vp1 (TaVp1), an orthologue of maize Vp1, and found it to be located 30 cM from the R gene. Another Myb-type transcription factor of maize, P, which regulates the expression of CHS, CHI, and DFR, leads specifically to the production of phlobaphene (Grotewold et al., 1994). Bailey et al. (1999b) located one of the two wheat P orthologues on a chromosome of homoeologous group 4.
So far there is no information on the genes for enzymes or regulatory factors in the flavonoid biosynthesis pathway of wheat. DFR is located in an important regulatory point in the pathway and is upstream of anthocyanin, proanthocyanidin, and phlobaphene production (Winkel-Shirley, 2001; Polashock et al., 2002).
Three wheat DFRs located on chromosomes 3A, 3B, and 3D have been cloned. Nucleotide sequences in the promoter region of these DFRs included two or three combinations of a G box core element and a binding site for a Myb-type transcription factor, P, but were different among the DFRs. They might function differently in wheat tissues since the three DFRs were expressed at different levels in grains, leaves, and roots.
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Materials and methods |
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Plant materials
Wheat lines with different grain colours and different chromosomal constitutions of homoeologous group 3 (Table 1) were grown under a semi-transparent plastic roof in a field. The spikes of Triticum aestivum cv. Chinese Spring (CS, red-grained) and Novosibirskaya 67 (NS 67, white-grained) were tagged at anthesis and harvested at 5 d intervals from anthesis to 30 DPA. Grains were collected from the primary and secondary florets of the central spikelets of spikes. For DNA and RNA preparation, developing grains and seedlings grown at 20 °C under 12 h of UV light (about 100 mol m–2 s–1, UV lamp) or darkness were used.
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DNA and RNA isolation
DNA was isolated from 1 g of 10-d-old seedlings of CS and Norin 61 according to Murray and Thompson (1980). Total RNA was extracted from about 0.5 g of whole grains of CS and NS 67 collected at 5 DPA, the coat tissues of grains collected 10–30 DPA, and 1 g of leaves and roots of 3-d-old seedlings by the SDS–phenol method (Kawakami et al., 1992). The grain coat tissue was separated from embryo and endosperm with stainless steel micro spatulas. The tissues were frozen with liquid nitrogen and stored at –80 °C immediately after collection. Poly (A)+ RNA was isolated from 10 mg of total RNA with an mRNA isolation kit (Roche Diagnostics, Mannheim, Germany), according to the instructions from the supplier.
Isolation of partial wheat DFR genes
Primers for DFR (A1-LP and DFR2-RP) were designed based on the genomic sequences of barley DFR (Kristiansen and Rohde, 1991) and maize DFR (Schwarz-Sommer et al., 1987) (Table 2; and see Fig. 2a of the Results). Partial sequences of wheat DFR were amplified by PCR in the GeneAmp PCR system 2400 (Perkin Elmer Life and Analytical Sciences Inc., Boston, MA). Genomic DNA (50 ng) of Norin 61 or CS was used as a template in a 50 µl reaction solution: 1x Ex Taq buffer (Takara, Japan), 0.2 mM dNTP mix (Takara), 0.5 µM of left primer and right primer, 10% glycerol, and 2.5 U/100 µl Ex Taq (Takara). The PCR conditions were as follows: 5 min denaturation at 94 °C, then 30 cycles of 1 min at 94 °C, 1 min at 58 °C, and 1 min at 72 °C. PCR products were cut out from the gel with a surgical knife after electrophoresis in Mupid (Cosmobio, Japan). PCR products were cloned into a T-vector produced from the pBluescript SK+ vector.
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The DNA sequence was determined by the ABI 310 sequencer (Perkin Elmer). The sequence was analysed using ClustalW and T-COFFEE software (Notredame et al., 2000). After sequencing the partial wheat DFR gene, new primers (DFRAB-LP, DFRD-LP, DFRAsp-RP, DFRBsp-RP, DFRD-RP, and DFRAB-RP) were designed in introns 1 and 2 of DFR (Table 2). These primers were used to differentiate three wheat DFRs.
The 3' and 5' regions of DFR cDNA
The 3' region of DFR cDNA was determined by the 3' RACE method. The first strand cDNA was synthesized from poly (A)+ RNA of CS grains collected at 5 DPA with 0.5 g of Oligo (dT)15 plus 3' adapter primer (Table 2) and reverse transcriptase (Superscript II, GIBCO-BRL, Life Technologies Inc., Rockville, MD, USA). After cDNA synthesis, RNA was digested by RNase H (Takara). The 3' region of DFR cDNA was amplified with DFR2-LP (Table 2) and the 3' adapter primers, cloned into T-vector from pBluescript SK+ vector (clone No. 1) and sequenced. To amplify the 3' region of DFR in genomic DNA, a DFR-3UTR primer (Table 2) in the 3' UTR region was designed.
The 5' RACE system (version 2.0, GIBCO-BRL) with a DFR2-RP primer was used to synthesize the first strand cDNA. After the first strand cDNA synthesis, RNA was digested. Unincorporated dNTPs, the primer and proteins were removed by spin cartridge (GIBCO-BRL). A homopolymeric tail was then added to the 3'-end of the cDNA using TdT and dCTP. The 5' region of DFR was amplified with DFR1-RP and 5'RACE primers (Table 2). The PCR products were cloned into the T-vector from pBluescript SK+ vector and sequenced.
Inverse PCR
Norin 61 genomic DNA (1.5 µg) was digested with 15 U of a restriction enzyme, BclI. DNA was ligated using a ligation high solution (Toyobo, Japan) and used as a template for PCR.
The 5' upstream region of DFR was first amplified by PCR in 20 µl of reaction solution with 30 ng of ligated DNA, 0.5 µM of TaDFR-LR and TaDFR-IP primers, and then amplified with TaA1-R and TaDFR-RR primers (Table 2). Three fragments (2.3 kb, 2.1 kb, and 1.0 kb) were cloned and sequenced. Based on these sequences, three primers, ProA-LP, ProB-LP, and ProD-LP that are specific for 5' regions of TaDFR-A, -B, and -D were designed (Table 2). The 5' upstream regions of CS DFRs were amplified with these primers and with the TaA1-R primer (see Fig. 2a in the Results). Transcription factor binding sites were searched for using the MOTIF program (Bioinformatic Center, Institute for Chemical Research, Kyoto University, available from the web site at http://motif.genome.ad.jp).
Southern analysis
Genomic DNA of Norin 61 and CS was digested with restriction enzymes, BglII, EcoRI, XbaI, and XhoI, separated on 0.7% (w/v) agarose gel, and transferred onto the nylon membrane Hybond-N+ (Amersham Pharmacia Biotech, Japan). The membranes were prehybridized in a solution of 50% formamide, 5x SSC (0.75 M NaCl, 75 mM tri-sodium citrate dihydrate; pH 7.5), 0.1% (w/v) N-lauroylsarcosin, 0.02% SDS, and 2% blocking reagent (Roche Diagnostics) at 42 °C and hybridized with a solution containing DIG-labelled clone No. 1 for 16 h. Clone No 1 was DIG-labelled by a PCR DIG Labeling Mix (Roche Diagnostics). The membranes were washed with a solution of 0.5x SSC and 0.1% SDS at 65 °C.
RT-PCR
The first-strand cDNA was synthesized in 200 ng of poly (A)+ RNA with 0.5 mg of Oligo (dT)15 and reverse transcriptase (Superscript II, GIBCO-BRL). An aliquot of 0.2 µl of the first-strand cDNA was added to a 20 µl PCR solution and amplified with TaDFR-LP and DFR2-RP primers (Table 2). The reaction was carried out under the following conditions: 2 min denaturation at 94 °C; then 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C.
For quantitative RT-PCR assays, PCR was performed with 25, 30, and 35 cycles to ensure that amplifications were within the linear range. Since DFR increased linearly within a range of these cycles, 35 cycles was chosen for PCR. The total amount of cDNA in the samples was also standardized after the amount of ubiquitin mRNA was evaluated with two primers, Ubi-LP and Ubi-RP (Joshi et al., 1991; Table 2). These ubiquitin primers were also used to evaluate DNA contamination in the poly (A)+ RNA samples before the first-strand cDNA synthesis as a negative control. Each of three DFRs was amplified with the following specific primers: TaDFRAsp-RP2 for TaDFR-A, TaDFRBsp-LP2 for TaDFR-B, and TaDFRDsp-RP2 for TaDFR-D designed in 3', 5' UTR and exon (Table 2).
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Results |
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Copy number and chromosomal location of DFR
The copy number of the DFR gene was estimated in the genomic DNAs of wheat lines (CS, ditelo 3DL, ditelo 3DS, N3AT3D, N3BT3D, N3DT3A, and N3DT3B) by a Southern blot analysis (Fig. 1). Three fragments (sizes: 13.3 kb, 9.3 kb, and 3.7 kb) were detected in the DNA digested with BglII of CS and ditelo 3DL. Three similar-sized DFR fragments were also observed in Norin 61 DNA digested with the other restriction enzymes, EcoRI, XbaI, and XhoI (data not shown). The 13.3 kb fragment was missing in ditelo 3DS, which lacked the long arm of chromosome 3D, and in N3DT3A and N3DT3B, which lost a pair of chromosome 3D (Fig. 1). The 13.3 kb fragment appears to be located on the long arm of chromosome 3D. The 9.3 kb and 3.7 kb fragments were also missing in N3BT3D and N3AT3D, which lacked chromosome 3B and chromosome 3A, respectively. These results showed that wheat DFRs were located on chromosomes 3A, 3B, and 3D, which were designated as TaDFR-A, TaDFR-B, and TaDFR-D, respectively.
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Differentiation of three wheat DFRs
DFR fragments of two sizes (about 950 bp and 890 bp) were amplified in the genomic DNA of CS and its aneuploids with A1-LP and DFR2-RP primers designed based on the DFR nucleotide sequences of maize and barley (Figs 2a, 3a). The 890 bp fragment was not amplified in N3AT3D, and the 950 bp fragment was not amplified in N3DT3A. The fragments of 890 bp and 950 bp appear to be a part of TaDFR-A and TaDFR-D, respectively. At this point it was not possible to differentiate TaDFR-B from either of the two fragments. These two fragments were cloned and sequenced. Nucleotide sequences of these fragments showed high similarity with that of barley DFR and included three putative introns.
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Positions of these introns were confirmed in the cDNA sequence amplified by the 3' and 5' race methods and placed in Fig. 2a. Three introns were located at the same positions as those reported in the barley DFR gene. The DFRD-LP and DFRD-RP primers were designed in the first and second introns of the 950 bp fragment (Fig. 2a). A 280 bp fragment was amplified with these primers in N3AT3D and N3BT3D, but not in N3DT3A (Fig. 3b). The results confirmed that DFRD-LP and DFRD-RP primers specifically amplified a part of TaDFR-D and that the 950 bp fragment was a partial sequence of TaDFR-D. Primers (DFRAB-LP and DFRAB-RP) were designed in the first and second introns of the 890 bp fragment (Fig. 2a). Using these primers, two different fragments (about 290 bp and 270 bp) were amplified in N3AT3D and N3BT3D, suggesting that TaDFR-A and -B differ in the size of their introns (data not shown). After the sequencing of these fragments, DFRAsp-RP and DFRBsp-RP primers were newly designed in the second intron (Fig. 2a). A fragment of approximately 270 bp was amplified with a pair of DFRAB-LP and DFRAsp-RP primers in N3BT3D and N3DT3A, but not in N3AT3D (Fig. 3b). Also, a fragment of about 260 bp was amplified with a pair of DFRAB-LP and DFRBsp-RP primers in N3AT3D and N3DT3A, but not in N3BT3D (Fig. 3b). These results confirmed the sequences of the first and second introns of TaDFR-A and TaDFR-B.
The 3' and 5' regions of DFR
The 3' region of DFR cDNA was amplified with DFR2-LP designed based on the barley DFR sequence and Oligo (dT)15 with the 3' adapter primers and was sequenced. The DFR-3UTR primer was designed in the 3' UTR region (Fig. 2a). The 3' region of TaDFR-D was amplified by a pair of DFRD-LP and DFR-3UTR primers in CS, and the 3' region of TaDFR-A and TaDFR-B by a pair of DFRAB-LP and DFR-3UTR primers in N3BT3D and N3AT3D, respectively. Nucleotide sequences of these PCR products also confirmed the intron sequences of TaDFR-A, -B, and -D. Sequences of these fragments were included in the DFR sequences in Fig. 2a.
The 5' regions of the DFRs were analysed by the inverse PCR method. One site was cut by BclI in the nucleotide sequence between the primer sites of A1-LP and DFR-3UTR (Fig. 2a). Norin 61 DNA was digested with BclI and ligated. The 5' regions of the DFRs were amplified with the primers TaA1-R and TaDFR-RR (Fig. 2a). Three fragments (2.5 kb, 2.3 kb, and 1.2 kb) were obtained and their sequences were classified based on the sequences of the second introns of TaDFR-A, TaDFR-B, and TaDFR-D. Three primers (ProA-LP, ProB-LP, and ProD-LP) were designed in the 5' regions (Fig. 2b). The 5' upstream regions of CS DFRs were amplified by these primers and TaA1-R primer (Fig. 2). Nucleotide sequences of the 5' regions of three CS DFRs were shown in Fig. 2b. A transcription initiation site of wheat DFR, estimated by the longest sequence obtained by the 5' RACE method, was at the same site as that of barley DFR (Fig. 2a).
Similarity of DFR and transcription element in the promoter
The deduced amino acid sequences estimated for the TaDFR-A, TaDFR-B, and TaDFR-D genes were highly similar to each other and to those reported for DFRs of barley, rice, and maize (Kristiansen and Rohde, 1991, GenBank accession number S69616
[GenBank]
; Nakai et al., 1998, GenBank accession number AB003496
[GenBank]
; Schwarz-Sommer et al., 1987, GenBank accession number X05068
[GenBank]
) (data not shown). The nucleotide sequence similarity was also maintained to a certain degree in the first and third introns, and 3' and 5' untranslated regions among wheat DFRs (Fig. 2).
In the 5' upstream region of TaDFR-A, TaDFR-B, and TaDFR-D, several elements to which transcription factors can bind were found. Hartmann et al. (1998) found a light-responsive unit (LRU) in the CHS promoter region of Arabidopsis, which included two functional elements: a G-box core element (ACGT) and a Myb recognition element. Two combinations of the G box core element (ACGT) and a Myb-like transcription factor, P, binding site (ACCWACCNN, Grotewold et al., 1994) were found in the promoter of TaDFR-A and TaDFR-D and three combinations in TaDFR-B (Fig. 2b). Two core elements (AAAG) for Dof transcription factor (one zinc finger), which has been reported to be involved in tissue specificity and light regulation (Yanagisawa and Schmidt, 1999), were in all three promoters. A TACpyAT element (van der Meer et al., 1992) and a core element for SBF-1 factor (GGTTAA, Green et al., 1988; Lawton et al., 1991), which was responsible for organ-specific expression, were in all three promoters and in the promoter of TaDFR-A, respectively (Fig. 2b).
Chromosomal location of wheat DFRs
To compare the location of DFR with that of the R gene on the long arm of wheat chromosome 3, TaDFR-A, TaDFR-B, and TaDFR-D were amplified in the partial deletion lines of chromosomes 3AL (3AL-1, 3AL-3), 3BL (3BL-4 and 3BL-5), and 3DL (3DL-1 and 3DL-2) with ditelos 3AL, 3BL, and 3DL. Primers of TaDFR-LP, DFR1-RP, TaDFRAsp-RP2, TaDFRBsp-LP2, and TaDFRDsp-RP2 were newly designed in the exons of three DFRs (Fig. 2) and used to amplify each DFR in the following combinations: TaDFR-LP and TaDFRAsp-RP2 for TaDFR-A, TaDFRBsp-LP2 and DFR1-RP for TaDFR-B, and TaDFR-LP and TaDFRDsp-RP2 for TaDFR-D. These primers were also used later in the expression assay of each DFR in different wheat tissues.
TaDFR-A was amplified in 3AL-3, but not in 3AL-1 (Fig. 4a). TaDFR-B and TaDFR-D were also detected in 3BL-5 and 3DL-2, respectively, not in 3BL-4 and 3DL1 (Fig. 4b, c). These results showed that DFR located in the proximal region of chromosomes of homoeologous group 3.
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Partial deletion lines of the long arm of chromosome 3D (3DL-1 and 3DL-2) lacked the functional R-D1b allele, showing a white grain colour, while TaDFR-D was located between the break points of 3DL-2 and 3DL-1. The results showed that the R gene was different from TaDFR. Compared with molecular maps of the long arm of chromosome 3 (McGuire and Qualset, 1997; Bailey et al., 1999a), TaDFR-D was located in a more proximal region than the taVp1 and R genes in the long arm of chromosome 3D (Fig. 4d).
Expression of DFR gene
Grain colour development of CS was examined from 5–30 DPA (Fig. 5). Grains from 5–10 DPA were pale blue, while those at 15 DPA were green. At 20 DPA, the green colour had faded. From 25 DPA, grains became yellow and thereafter turned brown. The red pigment in the grain coat appeared to develop before 20 DPA. Expression of the DFR gene was examined in whole grains at 5 DPA and in the grain coat tissue from 10–30 DPA of CS by the RT-PCR method with a pair of TaDFR-LP and DFR2-RP primers (Fig. 5). DFR expression was high in the whole grains at 5 DPA and in grain coat tissue at 10 DPA, while it gradually decreased from 15 to 20 DPA in CS. On the other hand, DFR was expressed at extremely low level in the white-grained line, NS 67, during the early stage of grain development (Fig. 5).
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The expression levels of three DFRs were compared in grains, leaves, and roots of CS. TaDFR-B and -D were expressed more than TaDFR-A in grains (Fig. 6). In leaves collected from 3-d-old seedlings of CS grown at 20 °C in darkness or under 12 h of UV light, DFRs were activated more in the light, while no light stimulation was observed in roots. In leaves and roots, TaDFR-B was expressed more than the other two.
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Discussion |
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Hexaploid wheat has three DFR genes; one each on chromosomes 3A, 3B, and 3D. Partial deletion lines of chromosome arm 3AL, 3BL, and 3DL showed that the TaDFRs were located in a more proximal region of the long arm than R and TaVp1. The DFR locus corresponded to that of maize DFR on the proximal region of the long arm of maize chromosome 3 as reported by Bailey et al. (1999a). Unpublished results from the authors’ laboratory showed that CHS, CHI, and F3H, required for the production of phlobaphene and proanthocyanidin, were not located on chromosome 3. The R gene might not be a structural gene for the enzymes in the flavonoid synthesis pathway.
DFR was expressed more in the grain coat tissue of CS (red-grained) than in that of the white-grained line, NS 67, during early grain development (5–15 DPA). The R gene might be involved in the expression of DFR. The time lag between DFR expression and grain colour change observed from 20 DPA might reflect the process of phlobaphene or proanthocyanidin production after a DFR step. CHS, CHI, and F3H were also expressed in the seed coat tissue of red-grained wheat, but not in that of white-grained wheat (E Himi, K Noda, unpublished data). These results give further support to the R gene being a transcription factor for flavonoid biosynthesis.
A Myb-like transcription factor, P, recognizes the CCT/AACC element in the promoter of maize DFR and activates CHS, CHI, and DFR genes in the flavonoid biosynthesis pathway leading to the production of phlobaphene in maize (Grotewold et al., 1994, 1998; Chopra et al., 1996). Sainz et al. (1997) found that P and another Myb-type transcription factor, C1, could bind to the CCT/AACC element and sequences similar to CCT/AACC. Wheat DFRs have three putative P-binding sites in their promoters (Fig. 2b). Rice and barley also have one or two putative P-binding sites in the 5' upstream regions of DFR (Nakai et al., 1998; Kristiansen and Rohde, 1991). A P-like transcription factor appears to be a key transcription factor for activation of wheat DFRs.
The differences found in the promoter regions of the three wheat DFRs make it possible to compare the promoter function of the DFRs. The three DFRs were expressed differently in grains (Fig. 6). TaDFR-B and -D were up-regulated more in the grains than TaDFR-A. TaDFR-B carried three combinations of a G-box element and a myb-binding element in its promoter, while TaDFR-A and TaDFR-D had two combinations of these elements. In Arabidopsis, parsley, and mustard this combination of cis-elements was involved in light-responsive and tissue-specific expression of CHS (Kaiser and Batschauer, 1995; Hartmann et al., 1998; Schulze-Lefert et al., 1989). Three combinations of these elements in TaDFR-B might be responsible for the high expression of TaDFR-B. The SBF-1 factor has been reported to act as a transcriptional silencer for organ-specific expression of CHS (Lawton et al., 1991). The SBF-1 element exists only in the TaDFR-A promoter. It may cause the low level expression of TaDFR-A in all the tissues examined: grains, leaves, and roots. In leaves, three DFRs responded to light (Fig. 6). In addition to the combinations of a G-box element and a myb-binding element, two Dof elements which are also responsive to light-regulated and tissue-specific gene expression (Yanagisawa and Sheen, 1998) were found in the promoters. Hartmann et al. (1998) suggested there was an additional functional sequence to LRU in the CHS promoter for activating CHS. The Dof element will be a candidate for the light-responsive element in DFR. In roots, TaDFR-D expression was lower than TaDFR-B expression. van der Meer et al. (1992) showed that a TACPyAT element was responsible for the organ-specific expression of CHS. However, this element was detected in all three promoters and can not explain the difference of DFR expression in roots.
The present results for three wheat DFRs suggest that these DFRs differentiated in their promoter regions during evolution and came to share the expression of DFR differently depending on tissues.
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Acknowledgements |
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We thank Drs Masahiko Maekawa, Shigeko Utsugi, and Kazuhide Rikiishi for helpful discussions. We also express our appreciation to Dr Takashi R. Endo, Kyoto University, Japan, and Dr John Flintham, John Innes Centre, Norwich, UK, for providing the seeds of deletion lines, and Novosibirskaya 67. This work was supported in part by grants from the Ohara Foundation for Agricultural Research and the Ministry of Agriculture.
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