SEARCH

SEARCH BY CITATION

Keywords:

  • dihydroflavonol 4-reductase;
  • pelargonidin;
  • anthocyanin;
  • substrate specificity;
  • site-directed mutagenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4-reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin-type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate-binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Anthocyanins are major pigments which, together with carotenoids, are responsible for flower color. Three different classes of anthocyanidins are responsible for the primary shade of the flower color: pelargonidin (orange to brick red), cyanidin (red to pink) and delphinidin (purple to blue) (Tanaka et al., 1998). Biosynthetic pathways for the anthocyanins are fairly well established (Dixon and Steele, 1999; Holton and Cornish, 1995; Shirley et al., 1995; Shirley, 1996). One of the biosynthetic enzymes, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of three colorless dihydroflavonols – dihydrokaempferol (DHK), dihydroquercetin (DHQ) and dihydromyricetin (DHM) – to leucoanthocyanidins. These are subsequently converted to pelargonidin, cyanidin and delphinidin (Figure 1). The three substrates of DFR are very similar in structure, differing only in the number of hydroxyl groups on the B phenyl ring, which is not the site of enzymatic action. Thus it is not surprising that DFRs from many species can utilize all three substrates (Helariutta et al., 1993; Heller et al., 1985; Meyer et al., 1987; Stich et al., 1992; Tanaka et al., 1995).

image

Figure 1. A schematic diagram showing the chemical reaction catalyzed by dihydroflavonol 4-reductase.

Abbreviations used are; CHS: chalcone synthase, CHI: chalcone isomerase, F3H: flavanone 3-hydroxylase, F3′H: flavonoid 3′-hydroxylase, F3′5′H: flavonoid 3′5′-hydroxylase, DFR: dihydroflavonol 4-reductase, ANS: anthocyanin synthase, 3GT: flavonoid 3-glucosyltransferase.

Download figure to PowerPoint

Since most DFRs can utilize all three dihydroflavonols as substrates, the synthesis of the three different anthocyanidins are mainly determined by the enzyme activities of two hydroxylases: flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). F3′H converts DHK to DHQ and F3′5′H converts DHK to DHM or DHQ to DHM (Brugliera et al., 1999; Holton et al., 1993). DFR competes for the dihydroflavonols with flavonol synthase and the hydroxylases to synthesize corresponding leucoanthocyanidins, precursors of anthocyanidins (Winkel-Shirley, 1999). Flower color is determined by the ratio of these anthocyanidins and subsequent modifications to the anthocyanin structure such as acylation, glycosylation and methylation. Further variation of color by these anthocyanins can be achieved by vacuolar pH and copigmentation. Therefore, F3′H and F3′5′H are important determinants of flower color in most plant species (Holton and Tanaka, 1994; Mol et al., 1998; Mol et al., 1999). However, DFRs from some species such as Petunia and Cymbidium cannot reduce DHK efficiently, thus these species cannot produce pelargonidin-based orange flower color even if both F3′H and F3′5′H are absent. (Forkmann and Ruhnau, 1987; Gerats et al., 1982; Johnson et al., 1999). The inability of Petunia DFR to reduce DHK was successfully overcome by the introduction of maize DFR to generate the orange flower color (Meyer et al., 1987).

It is not known how substrate specificity is determined in DFR, nor how common it is for DFRs to have altered substrate specificity in nature. An insightful hypothesis on a region determining substrate specificity was proposed based on the amino acid sequence alignment with DFR from Petunia, maize and Antirrhinum (Beld et al., 1989). The alignment indicated the presence of a variable region in the middle of well-conserved regions. This variable region was suggested to be a region that determines the substrate specificity of DFR. The experimental data supporting the hypothesis, however, were lacking.

Our goal was to identify the region that determines the substrate specificity of DFR and to modulate the substrate specificity by altering amino acids in that region. We generated chimeric DFRs using Petunia DFR that cannot reduce DHK and Gerbera DFR that can reduce DHK (Elomaa et al., 1995). By introducing the chimeric DFRs to the mutant Petunia line, we experimentally identified a region that determines the substrate specificity. Furthermore, we show that the substrate specificity of DFR can be modulated by altering amino acids in the region.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Identification of a region that determines the substrate specificity of DFR

In order to identify the region of DFR that determines its substrate specificity, we constructed chimeric DFR genes using cDNA sequences of Petunia and Gerbera. Although these two DFRs have high similarity at the amino acid level, Gerbera DFR is able to reduce dihydrokaempferol (DHK) while Petunia DFR cannot. We built three different chimeric genes using regions of high homology as common PCR primer sites (Figure 2a). The chimeric genes were transformed into a white flowered Petunia mutant (W80) that lacks DFR activity in flower and accumulates primarily DHK but which has appreciable amounts of dihydroquercetin (DHQ) and dihydromyricetin (DHM) (Huits et al., 1994). We chose the transgenic approach instead of biochemical assay using recombinant proteins because it provides data showing the substrate specificity of DFR in its native condition and it has been known that purified DFR enzyme is very unstable (Forkmann and Ruhnau, 1987). The introduction of full-length GerberaDFR produced flowers of an orange/brick-red color as reported previously (Elomaa et al., 1995; Johnson et al., 1999). Plants containing chimera 1 produced pink flowers while plants transformed with chimeras 2 and 3 bore orange/brick-red flowers (Figure 2b). The hue of transgenic flowers containing chimera 1 is very similar to the inbred Petunia mutant RLO1, which is similar to W80 line except for a functional DFR gene. Thin layer chromatography (TLC) determined that plants containing chimera 1 produced mainly cyanidin and delphinidin (Figure 2c), while plants transformed with chimeras 2 and 3 primarily produced pelargonidin (Figure 2c). These results indicated that the region of DFR conferring the ability to reduce DHK was located in the sequence that is present in chimera 2 but not in chimera 1. The identified region (approximately 40 amino acids) is highly variable in DFRs from different plant species. By excluding the completely conserved amino acid sequences at the borders, the identified region is narrowed down to 26 amino acids (boxed in Figure 2d). The 26 amino acid region includes the previously suggested substrate specificity determining region (Beld et al., 1989). Alignment of 4 dicot DFRs and a maize DFR that are known to accept DHK as a substrate and Petunia DFR shows that 4 out of 26 amino acids are completely conserved (shown in bold in Figure 2d). Comparison indicates that 10 amino acids are different between Petunia and Gerbera DFR in this region and 4 residues are conserved in other DFRs but not in Petunia DFR (marked by an * in Figure 2d). The 4 residues, however, are not conserved between Petunia and Cymbidium DFRs, suggesting that different amino acids determine the substrate specificity of these two enzymes. If we compare the codons of 4 residues that are conserved in DHK-accepting DFRs but not in Petunia DFR, alteration of a single nucleotide is sufficient to convert an amino acid of Petunia DFR to the corresponding amino acid of DHK-accepting DFRs. If a single nucleotide change is required to convert Petunia DFR to DHK-accepting DFR, a Petunia line that produces orange-colored flowers could have been found in nature or been developed by the conventional breeding programs. Based on this, we reasoned that alteration of more than one amino acid may be required to convert Petunia DFR to Gerbera-like DFR.

image

Figure 2. Identification of a region that determines the substrate specificity of DFR.

(a) C.1, C.2 and C.3 indicate different chimeric DFRs. Chimeric DFRs were constructed using Gerbera DFR and Petunia DFR. Black bars indicate the portion of DFR originating from Gerbera DFR and grey bars indicate the portion of DFR originating from Petunia DFR. Numbers at the junctions are the amino acid numbers in Gerbera DFR. Petunia DFR is 381 amino acids long while Gerbera DFR is 366 amino acids long.

(b) Representative flowers of transgenic Petunia expressing chimeric DFRs or control DFRs. Ger indicate the transgenic flower expressing Gerbera DFR and C.1, C.2, and C.3 indicate those expressing different chimeric DFRs. RL01 line has a functional Petunia DFR gene. The C.1 and RL01 bore similar pink colored flowers while others bore brick-red colored flower.

(c) TLC analysis of pigments produced in the transgenic flowers. Pg, Cy and Dp indicate pelargonidin, cyanidin, and delphinidin pigments. The C.1 did not accumulate pelargonidin while the C.2 and C.3 accumulated pelargonidin like Ger.

(d) Alignment of 5 DHK-accepting DFR and Petunia DFR. The identified 26 amino acids are boxed. Amino acids that are conserved in all DFRs are bold-typed and amino acids that are different in Petunia DFR are marked with a *. Ger: Gerbera DFR, Rosa: Rose DFR, Antir: Antirrhinum DFR, Dian: Carnation DFR, Zea: Maize DFR, Pet: Petunia DFR.

Download figure to PowerPoint

Site-directed mutagenesis of the substrate specificity determining region

To prove that mutations in the identified 26 amino acid sequence can alter the substrate specificity of DFR, we performed in vitro mutagenesis on Gerbera DFR at some of the hydrophilic residues (Figure 3a). Since the three dihydroflavonol substrates differ only in hydroxyl groups of the B-ring, we reasoned that hydrogen bonding might play an important role in distinguishing the three dihydroflavonols. In a crystal structure of chalcone synthase (CHS), another flavonoid enzyme, hydrogen bonds between CHS and the hydroxyl groups of the B-ring of naringenin can be observed (Ferrer et al., 1999). After mutating each residue to a similar sized hydrophobic amino acid, we transformed each mutant GerberaDFR into the white-flowered Petunia mutant described above. All but two of the amino acid substitutions resulted in flowers that were colored exactly like the wild type GerberaDFR transformants (Figure 3b). The mutation E145L resulted in white flowers in all transformants. Sequence analysis was done to confirm that no other unintended mutation was introduced. In addition, transgenic DFR mRNA was detected in the primary E145L transformants by RT–PCR analysis (data not shown). These results indicate that the substitution of glutamate to leucine at this site abolished DFR enzyme activity. It is interesting to note that 145th glutamine is conserved in all DHK-accepting dicot DFR but not in Petunia DFR. Transformants containing the mutation N134L produced slightly different flower colors compared to wild type GerberaDFR or other mutated DFRs (Figure 3b). TLC analysis of flower pigments from the mutant N134L showed that corollas from this mutant only synthesized pelargonidin and not cyanidin or delphinidin (Figure 3c). This indicates that this asparagine residue may be a critical binding site of dihydroquercetin and dihydromyricetin. Alternatively, the substitution of leucine could have introduced a steric hindrance affecting the binding of these two substrates. Like 145th glutamine, 134th asparagine is conserved in DHK-accepting DFRs but not in Petunia DFR. Although the exact molecular mechanism of substrate specificity determination in N134L mutant is not known yet, the result shows that a mutation in a single amino acid in the region can alter the substrate specificity of DFR. The data further suggest that flower color in different flower species or in different cultivars of the same species can be influenced by relatively minor changes in this region of DFR.

image

Figure 3. Site-directed mutagenesis of the substrate specificity determining region.

(a) The sequence corresponds to the substrate specificity determining region of Gerbera DFR. Arrows and letters indicate the amino acid changes.

(b) Flowers of transgenic Petunia expressing mutated Gerbera DFR gene. Ger indicates the wild type Gerbera DFR and T132V indicates the mutated DFR that has valine instead of threonine at the 132nd position of Gerbera DFR. Names of other mutated DFRs followed the same notation rule. All transgenic lines except N134L and E145L have the same brick red colored flower. The N134L bore slightly different colored flowers and E145L bore white flowers.

(c) TLC analysis of pigments produced in the transgenic petunia flowers. As expected, the E145L did not accumulate any anthocyanin. The N134L accumulated mostly pelargonidin while other mutated DFR and wild type Gerbera DFR accumulated significant amounts of cyanidin and delphinidin in addition to pelargonidin.

Download figure to PowerPoint

Development of a DFR that preferentially utilizes DHK as a substrate

Since the W80 Petunia line used for transformation accumulates mainly DHK with small amounts of DHQ and DHM, it was not clear if the N134L mutant DFR completely lost the capability of reducing DHQ and DHM. To investigate if the N134L mutant DFR produces only pelargonidin in the presence of fully active flavonoid-3′-hydroxylase (F3′H) or flavonoid-3′,5′-hydroxylase (F3′5′H), we crossed our N134L transformant with Petunia lines that are either dfr–/–/F3′H+/+ (WR line) or dfr–/–/F3′5′H+/+ (WV line). As shown in Figure 4(a), both WR and WV lines produce white flowers. When these lines were crossed with the N134L transformants, the WR line expressing the mutant DFR (WR/DFRN134L) had orange colored flowers while the WR expressing wild type Gerbera DFR (WR/DFRGER) had red colored flowers. In contrast, the WV lines expressing the mutant DFR (WV/DFRN134L) bore white flowers while WV lines expressing the wild type DFR (WV/DFRGER) had violet colored flowers. To determine the pigments produced in these crossed lines, we performed TLC analysis. Figure 4(b) shows that the WR/DFRN134L accumulated a large amount of pelargonidin while WR/DFRGER mainly accumulated cyanidin. In the white flowered WV/DFRN134L, no appreciable amounts of anthocyanidins accumulated, while the WV/DFRGER accumulated mainly delphinidin. The data indicate that the N134L mutant DFR preferentially utilizes DHK as a substrate over DHQ and cannot efficiently reduce DHM. The substrate preference of the N134L mutant DFR is somewhat opposite to that of Petunia DFR which prefer DHM to DHQ and cannot use DHK (Forkmann and Ruhnau, 1987).

image

Figure 4. Development of a DFR that displays altered substrate specificity.

(a) WR and WV indicate petunia lines that are dfr–/–, but F3′H+/+ (WR) or F3′5′H+/+ (WV). The mark – indicates no DFR gene, DFRN134L indicates DFR that has leucine instead of asparagine at the 134th position of Gerbera DFR, and DFRGER indicates the wild type Gerbera DFR.

(b) TLC analysis of pigments produced in the transgenic lines. Pg, Cy and Dp indicate pelargonidin, cyanidin and delphinidin. The WR and WV lines expressing wild type DFR accumulated cyanidin and delphinidin each. The WR line expressing DFRN134L accumulated pelargonidin and cyanidin, while the WV line expressing DFRN134L did not accumulate any pigment other than background level of delphinidin.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Petunia DFR exhibits a peculiar substrate specificity (Forkmann and Ruhnau, 1987). Because Petunia DFR cannot reduce DHK, the precursor of orange pigment, the transformation of a maize DFR gene was used to generate orange colored Petunia flowers (Meyer et al., 1987). To determine the region of the DFR enzyme responsible for substrate specificity, we introduced chimeric DFR genes of Petunia and Gerbera into a DHK-accumulating Petunia mutant line. We identified a 26 amino acid region that overlaps with a previously suggested substrate specificity region based on an amino acid sequence alignment (Beld et al., 1989). Furthermore, we have shown that the substrate specificity of DFR can be relatively easily altered by developing a DFR that preferentially utilizes DHK as a substrate. Our results indicate that the reduction of dihydroflavonols by DFR can be an important enzymatic step for flower color determination in addition to the hydroxylation steps of DHK by F3′H or F3′5′H (Brugliera et al., 1999; Holton et al., 1993).

The variability of amino acid sequences in the region and the alteration of substrate specificity by a single mutation in the region suggest that more DFRs from different plant species or even in different cultivars of the same species can be proven to have different substrate preferences. DFRs with different substrate specificity can determine flower color as we have shown that the wild type Gerbera DFR specified violet flower color while the N134L mutant DFR specified white flower color in the presence of an active F3′5′H. Our results indicated that the mutant DFR could not accumulate pelargonidin in the presence of F3′5′H. It is not clear, however, why the DFRN134L could not accumulate pelargonidin, while DFRGER accumulated the expected delphinidin in WV line. It could be due to either higher enzyme activity of F3′5′H compared to that of DFRN134L or a proposed flavonoid biosynthetic enzyme complex (Burbulis and Winkel-Shirley, 1999), where DHK might be hydroxylated to DHM by F3′5′H before it is reduced by DFR. Further investigation is required to resolve this question.

The site directed mutation of 134th asparagine to leucine conferred Gerbera DFR to accept DHK preferentially as a substrate. This result suggests that 134th asparagine plays an important role in substrate specificity. Asparagine has an uncharged polar amide side chain that can potentially form hydrogen bonds. In contrast to asparagine, leucine has a non-polar side chain that cannot form hydrogen bonds. In addition to the difference of hydrophilicity, the side chain of leucine is much bulkier than that of asparagine. Thus it is possible that the substitution of asparagine to leucine could either disrupt the hydrogen bonding between the enzyme and the 5′-, or both 3′- and 5′-hydroxyl groups in the substrate or generate steric hindrance between the binding site and the hydroxyl groups. Although the substrate specificity of DFRN134L differs from that of Petunia DFR, it is interesting to note that 134th asparagine is conserved in most DFR while Petunia DFR has aspartic acid. Unlike asparagine, aspartic acid has a charged polar side chain with a pKA value of 3.9. Thus, aspartic acid will provide a more acidic surface that could potentially affect the binding of certain substrates. We are currently investigating if the conversion of asparagine to aspartic acid alone or with other mutations will change the substrate specificity of Gerbera DFR to that of Petunia DFR.

Another question that needs to be further investigated is how the identified region affects substrate specificity. A comparison of the predicted secondary structure of the region with a known crystal structure of a related enzyme might give a clue to the role of the region. DFR is a member of the enzyme superfamily called short chain dehydrogenase/reductase (SDR) that includes two lignin biosynthesis enzymes – cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase (Baker and Blasco, 1992; Goffner et al., 1998; Lacombe et al., 1997). Other members of this diverse family include bacterial (cholesterol dehydrogenase, UDP-galactose 4-epimerase) and mammalian enzymes (3-β-hydroxysteroid dehydrogenase) (Jornvall et al., 1995). Alignment of a number of these superfamily members revealed a highly conserved NAD binding site in the N-terminal region (Lacombe et al., 1997). Baker and Blasco (1992) proposed that a putative ancestor of DFR may have been derived from UDP-galactose 4-epimerase, whose structure has been solved (Thoden et al., 1996a; Thoden et al., 1996b; Thoden et al., 1997). Crystal structures of many other SDR family members also have been solved (Benach et al., 1998; Bennett et al., 1996; Duax et al., 1996; Hulsmeyer et al., 1998; Penning et al., 1997; Tanaka et al., 1996). Although epimerase and DFR catalyze different chemical reactions, the two enzymes are phylogenetically closely related, and thus we included only epimerase in Figure 5, which shows the comparison between DFR and other SDR family members. A serine residue and Tyr-X3-Lys couplet in the SDR family members have been shown to be critical for catalysis. Sequence alignment of a number of DFRs and SDR family members shows that a Ser and a Tyr-X3-Lys couplet adjacent to our putative substrate binding region are completely conserved in all DFRs (marked by * in Figure 5). In addition, the crystal structures of SDR family members and the predicted secondary structure of DFR around the Ser and Tyr-X3-Lys couplet have the similar β-sheet–loop–α-helix structure (Figure 5) (Thoden et al., 1996a; Thoden et al., 1996b; Thoden et al., 1997). However, DFR has a longer loop than the epimerase. The length difference of the loop may reflect the length of side chains between the two substrates: UDP-galactose has a hydroxyl group while dihydroflavonol has a hydrophobic phenyl group. Based on the data and sequence analysis, we propose that the substrate specificity-determining region we identified is a binding pocket for the dihydroflavonol B-ring, and the Ser and Tyr-X3-Lys couplet next to the region form a part of the active site of DFR. Further biochemical and structural analyzes will help to solve the role of this region for catalytic activity and the determination of the substrate specificity of DFR.

image

Figure 5. Sequence analysis of a region that determines substrate specificity.

Boxed amino acids indicate the substrate specificity determining region. Bold letters marked by * indicates the putative amino acids that form a part of active site in DFR and UDP-galactose epimerase superfamily. The 134th asparagine is marked by an arrowhead. Dots in the sequence indicate gaps. Jnet and Jpred are the results of secondary structure prediction and UGE str indicates the secondary structure found in a crystal of UDP-galactose epimerase. E (Extended, β-sheet), H (Helix, α-helix). Gerbera: Gerbera DFR, Petunia: Petunia DFR, Rosa: Rose DFR, Antirrh: Antirrhinum DFR, Dianth: Carnation DFR, Zea: Maize DFR, Eco UGE: UDP-galactose epimerase.

Download figure to PowerPoint

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Petunia transformation

Leaf explants of the inbred Petunia W80 line (an6, ht1, ht2, hf1, hf2, fland rt) were transformed as described elsewhere, except that leaf explants recently infected by Agrobacterium tumefaciens were rinsed with Murashige–Skoog solution containing 750 mg l−1 cefotaxime and then placed on media having 100 mg l−1 kanamycin sulfate and 500 mg l−1 cefotaxime (Johnson et al., 1999). Also, putative transformants were grown on MS media with vitamins, 30 g l−1 sucrose, 0.6% agar and 500 mg l−1 cefotaxime; after rooting the transformants were transferred to soil.

Chimeric gene construction

Highly conserved regions of the DFR gene were identified by a multiple sequence alignment of a number of DFRs. The 5′ region (Gerbera DFR portion) of each chimeric gene was synthesized from the Gerbera DFR cDNA clone using a primer containing the codon for the starting methionine of the Gerbera DFR gene (5′-GGC GAA AAT GGA AGA GGA TTC TCC-3′) and a primer containing a conserved region of the Gerbera DFR gene (Chimera 1: 5′-AGC AGA TGA AGT GAA CAC TAG TTT CTT CAC-3′; Chimera 2: 5′-GGC TTT CTC TGC CAG AGT TTT TGA CAC GAA-3′; Chimera 3: 5′-GTG GGA CGA GCA AAT GTA TCT TCC TTT TGC-3′). The 3′ region (Petunia DFR portion) of each chimeric gene was synthesized from the Petunia DFRA cDNA clone using a primer complementary to the three conserved regions (Chimera 1: 5′-TTC ACT TCA TCT GCT GGA ACT CTC GAT GTG; Chimera 2: 5′-CTG GCA GAG AAA GCC GCA ATG GAA GAA GCT-3′; Chimera 3: 5′-ATT TGC TCG TCC CAC CAT GCT ATC ATC TAC-3′) and a primer containing the stop codon of the Petunia DFRA gene (5′-GCG CTA GAC TTC AAC ATT GCT TAA-3′). 5′ and 3′ regions were gel purified after PCR amplification. To assemble the full length chimeric gene the 5′ and 3′ region fragments were added to the same tube in roughly equal amounts and subjected to 25 PCR cycles (94°C 30′, 55°C 30′, 72°C 1 : 30). Full length chimeric genes (∼1.1 kb) were purified from agarose gels. The chimeric genes were cloned into a vector containing the 35S CaMV promoter and NOS terminator. Pfu polymerase (Stratagene, La Jolla, CA, USA) was used for all PCR reactions.

Amino acid point mutant construction

Gerbera DFR genes containing one amino acid point mutation were made in a similar manner as the chimeric genes. The 5′ region was synthesized using a primer having the Gerbera DFR starting methionine and a primer containing a single codon change. The 3′ region was made with a complementary primer with the single codon change and a primer having the stop codon of Gerbera DFR. The full length mutant sequence was assembled like the chimeric genes above. Each point mutant was cloned into a vector having the 35S CaMV promoter and NOS terminator. The mutagenized region of each mutant DFR was sequenced to ensure the correct residue was changed. Point mutants were then transformed into the W80 Petunia line. The transformants expressing the DFR genes were crossed with WR Petunia line (dfr–/–, F3′H+/+) and WV Petunia line (dfr–/– F3′5′H+/+) to determine the substrate specificity of the mutated DFR. Mutations in other loci were not determined in these two Petunia lines.

TLC analysis

Anthocyanidins were separated on cellulose TLC plates as described previously (Johnson et al., 1999). Corollas were sometimes stored at 4°C for extended periods of time in methanol-0.5% HCl solution. Before adding iso-amylalcohol, the flower extracts were quantified at 530 nm to ensure uniform loading on the TLC plate. Anthocyanin standards were purchased from Apin Chemicals Ltd. (Oxfordshire, UK).

Sequence analysis

Secondary structure of aligned DFR sequences was predicted by the Jpred method (Cuff et al., 1998) and the secondary structure of E. coli UDP-Galactose epimerase found in a crystal structure was obtained based on the authors' description (Thoden et al., 1996a). Multiple sequence alignment was undertaken using Clustal W program (Thomson et al., 1994).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We are grateful to Dr Ronald Koes for providing W80 seeds, Dr Peter Meyer for giving RL01 seed, Dr Teemu Teeri for supplying the Gerbera DFR clone, and Dr Tom Gerats for supplying the Petunia DFR clone. We thank Dr Pill-Soon Song for his critical reading. The work is partially supported by Kumho Petrochemical Co. Ltd, KISTEP NRL program, KOSEF SRC program, and KOSEF Basic Research program.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Baker, M.E. & Blasco, R. (1992) Expansion of the mammalian 3 beta-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus. FEBS Lett. 301, 8993.
  • Beld, M., Martin, C., Huits, H., Stuitje, A.R. & Gerats, A.G. (1989) Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes. Plant Mol. Biol. 13, 491502.
  • Benach, J., Atrian, S., Gonzalez-Duarte, R. & Ladenstein, R. (1998). The refined crystal structure of Drosophila lebanonensis alcohol dehydrogenase at 1.9 A resolution. J. Mol. Biol. 282, 383399.DOI: 10.1006/jmbi.1998.2015
  • Bennett, M.J., Schlegel, B.P., Jez, J.M., Penning, T.M. & Lewis, M. (1996) Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. Biochemistry, 35, 1070210711.
  • Brugliera, F., Barri-Rewell, G., Holton, T.A. & Mason, J.G. (1999) Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant J. 19, 441451.DOI: 10.1046/j.1365-313x.1999.00539.x
  • Burbulis, I.E. & Winkel-Shirley, B. (1999) Interactions among enzymes of the Arabidopsis flavonoids biosynthetic pathway. Proc. Natl Acad. Sci. USA, 96, 1292912934.DOI: 10.1073/pnas.96.22.12929
  • Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M. & Barton, G.J. (1998) JPred: a consensus secondary structure prediction server. Bioinformatics, 14, 892893.DOI: 10.1093/bioinformatics/14.10.892
  • Dixon, R.A. & Steele, C.L. (1999) Flavonoids and isoflavonoids – a gold mine for metabolic engineering. Trends Plant Sci. 4, 394400.DOI: 10.1016/s1360-1385(99)01471-5
  • Duax, W.L., Griffin, J.F. & Ghosh, D. (1996) The fascinating complexities of steroid-binding enzymes. Curr. Opin. Struct. Biol. 6, 813823.
  • Elomaa, P., Helariutta, Y., Griesbach, R.J., Kotilainen, M., Seppanen, P. & Teeri, T.H. (1995) Transgene inactivation in Petunia hybrida is influenced by the properties of the foreign gene. Mol. Gen. Genet. 248, 649656.
  • Ferrer, J.L., Jez, J.M., Bowman, M.E., Dixon, R.A. & Noel, J.P. (1999) Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat. Struct. Biol. 6, 775784.DOI: 10.1038/11553
  • Forkmann, G. & Ruhnau, B. (1987) Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrida. Z. Naturforsch. 42, 11461148.
  • Gerats, A.G.M., Vlaming, P., Doodeman, M., Al, B. & Schram, A.W. (1982) Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida. Planta, 155, 364368.
  • Goffner, D., Van Doorsselaere, J., Yahiaoui, N., Samaj, J., Grima-Pettenati, J. & Boudet, A.M. (1998) A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression. Plant Mol. Biol. 36, 755765.
  • Helariutta, Y., Elomaa, P., Kotilainen, M., Seppanen, P. & Teeri, T.H. (1993) Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae). Plant Mol. Biol. 22, 183193.
  • Heller, W., Forkmann, G., Britsch, L. & Grisebach, H. (1985) Enzymatic reduction of (+) -dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis. Planta, 165, 284287.
  • Holton, T.A., Brugliera, F., Lester, D.R., Tanaka, Y., Hyland, C.D., Menting, J.G., Lu, C.Y., Farcy, E., Stevenson, T.W. & Cornish, E.C. (1993) Cloning and expression of cytochrome P450 genes controlling flower colour. Nature, 366, 276279.
  • Holton, T.A. & Cornish, E.C. (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell, 7, 10711083.
  • Holton, T.A. & Tanaka, Y. (1994) Blue roses – a pigment of our imagination? Trends Biotechnol. 12, 4042.
  • Huits, H.S., Gerats, A.G., Kreike, M.M., Mol, J.N. & Koes, R.E. (1994) Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida. Plant J. 6, 295310.
  • Hulsmeyer, M., Hecht, H.J., Niefind, K., Hofer, B., Eltis, L.D., Timmis, K.N. & Schomburg, D. (1998) Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution. Protein Sci. 7, 12861293.
  • Johnson, E.T., Yi, H., Shin, B., Oh, B.J., Cheong, H. & Choi, G. (1999) Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins. Plant J. 19, 8185.DOI: 10.1046/j.1365-313x.1999.00502.x
  • Jornvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J. & Ghosh, D. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry, 34, 60036013.
  • Lacombe, E., Hawkins, S., Van Doorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M. & Grima-Pettenati, J. (1997) Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J. 11, 429441.
  • Meyer, P., Heidmann, I., Forkmann, G. & Saedler, H. (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature, 330, 677678.
  • Mol, J., Cornish, E., Mason, J. & Koes, R. (1999) Novel coloured flowers. Curr. Opin. Biotechnol. 10, 198201.DOI: 10.1016/s0958-1669(99)80035-4
  • Mol, J., Grotewold, E. & Koes, R. (1998) How genes paint flowers and seeds. Trends Plant Sci. 3, 212217.DOI: 10.1016/s1360-1385(98)01242-4
  • Penning, T.M., Bennett, M.J., Smith-Hoog, S., Schlegel, B.P., Jez, J.M. & Lewis, M. (1997) Structure and function of 3 alpha-hydroxysteroid dehydrogenase. Steroids, 62, 101111.
  • Shirley, B.W. (1996) Flavonoids biosynthesis: new functions for an old pathway. Trends Plant Sci. 1, 377381.DOI: 10.1016/1360-1385(96)10040-6
  • Shirley, B.W., Kubasek, W.L., Storz, G., Bruggemann, E., Koornneef, M., Ausubel, F.M. & Goodman, H.M. (1995) Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J. 8, 659671.
  • Stich, K., Eidenberger, T., Wurst, F. & Forkman, G. (1992) Enzymatic conversion of dihydroflavonols to flavan-3′,4-diols using flower extracts of Dianthus caryophyllus L. (carnation). Planta, 187, 103108.
  • Tanaka, N., Nonaka, T., Tanabe, T., Yoshimoto, T., Tsuru, D. & Mitsui, Y. (1996) Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli. Biochemistry, 35, 77157730.
  • Tanaka, Y., Fukui, Y., Fukuchi-Mizutani, M., Holton, T.A., Higgins, E. & Kusumi, T. (1995) Molecular cloning and characterization of Rosa hybrida dihydroflavonol 4-reductase gene. Plant Cell Physiol. 36, 10231031.
  • Tanaka, Y., Tsuda, S. & Kusumi, T. (1998) Metabolic engineering to modify flower color. Plant Cell Physiol. 39, 11191126.
  • Thoden, J.B., Frey, P.A. & Holden, H.M. (1996a) Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli. Biochemistry, 35, 25572566.
  • Thoden, J.B., Frey, P.A. & Holden, H.M. (1996b) High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol. Protein Sci. 5, 21492161.
  • Thoden, J.B., Hegeman, A.D., Wesenberg, G., Chapeau, M.C., Frey, P.A. & Holden, H.M. (1997) Structural analysis of UDP-sugar binding to UDP-Galactose 4-epimerase from Escherichia coli. Biochemistry, 36, 62946304.
  • Thomson, J.D., Higgins, D.G. & Gibson, T.J. (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 46734680.
  • Winkel-Shirley, B. (1999) Macromolecular organization of the primary and secondary pathways of aromatic amino acid biosynthesis. Physiol. Plant. 107, 142149.