1. Introduction
Flower color influences the commercial value of ornamental plants. In general, floral coloration involves the accumulation of pigments such as flavonoids (including anthocyanins), carotenoids, and betalains. The flavonoid class anthocyanins are responsible for flower colors ranging from pale pink to blue in many ornamental plants including chrysanthemum (
Chrysanthemum morifolium), carnation (
Dianthus caryophyllus), lily (
Lilium longiflorum), gerbera (
Gerbera hybrida), petunia (
Petunia hybrida), and rose (
Rosa hybrida). The mechanism of anthocyanin biosynthesis has been extensively investigated, and most structural and regulatory genes involved in this process have been identified in several plant species [
1,
2,
3]. Dihydroflavonol 4-reductase (DFR) plays an important role in the formation of anthocyanins and proanthocyanidins, as shown in the
Figure 1. DFR catalyzes the conversion of the colorless dihydroflavonols, including dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM), into leucoanthocyanidins. These three colorless dihydroflavonols are substrates of flavonol biosynthesis that are catalyzed by flavonol synthase (FLS). Competition between FLS and DFR can modify the metabolic flux and alter flower color [
4,
5,
6].
Several studies have revealed that the substrate specificity of DFR plays an important role in the determination of anthocyanin types [
7,
8,
9,
10]. According to differences at the 134th amino acid residues in their substrate binding domains, DFRs are divided into three types, the asparagine (Asn)-type, aspartic acid (Asp)-type, and non-Asn/Asp-type. Previous studies have reported that Asn-type DFRs catalyze the conversion of all three dihydroflavonols (DHK, DHQ, and DHM), whereas Asp-type DFRs are highly specific for DHQ and DHM rather than for DHK as a substrate [
8,
9,
10]. However, not all Asn-type DFRs can catalyze all three dihydroflavonols as substrates. DFRs from sweet potato (IbDFR) and from freesia (FeDFR2) are classified into the Asn-type DFR, due to their 134th amino acid residues [
10,
11]. IbDFR catalyzes only the DHK but not DHQ and DHM, while FeDFR2 utilizes both the DHQ and DHM but not the DHK as a substrate. Other studies have shown that the neighboring residues of the substrate binding sites and NADP binding regions of DFRs play pivotal roles in protein activity in buckwheat (
Fagopyrum esculentum), gerbera, and grape hyacinth (
Muscari armeniacum) [
11,
12,
13]. These results imply that other factors can influence the substrate specificity of DFR enzyme.
Chrysanthemum, a perennial herbaceous flowering plant, is an important floricultural crop worldwide. The ray florets of chrysanthemum flowers can be pink, red, yellow, white, purple, green, or various bicolored forms due to the accumulation of different proportions of two pigments, i.e., anthocyanins and carotenoids [
14]. There are three basic types of anthocyanidins, namely, pelargonidin, cyanidin, and delphinidin. Although the ray florets of chrysanthemum only accumulate the cyanidin derivatives, the substrate feeding assay revealed that CmDFR could utilize DHK and DHM as a substrate for pelargonidin and delphinidin biosynthesis, respectively [
14,
15,
16].
In the current study, we identified two DFR genes, CmDFR-OB and CmDFR-RM, with different C-terminus, from chrysanthemum flowers with white and red colors, respectively, and demonstrated that these genes function in anthocyanin biosynthesis. We performed an in vitro enzyme assay with recombinant CmDFRs and verified their catalytic activities for different dihydroflavonol substrates. In a complementation assay with the Arabidopsis thaliana mutant transparent testa3-1 (tt3-1), CmDFR-RM restored anthocyanin accumulation in rosette leaves and proanthocyanidin accumulation in the seeds, but CmDFR-OB did not. Our study indicates that the CmDFRs play a functional role in anthocyanin biosynthesis and pigmentation in the ray florets of chrysanthemum.
3. Discussion
DFR is a NADPH-dependent reductase that converts dihydroflavonols such as DHK, DHQ, and DHM into leucoanthocyanidins such as leucopelargonidin, leucocyanidin, and leucodelphinidin, respectively. DFR proteins contain the highly conserved NADP(H) binding domain “VTGAAGFIGSWLIMRLLERGY” and a substrate binding domain. The latter domain is divided into three types including the Asn type, Asp type, and non-Asn/Asp type, which differ in the amino acid residue at the 134th position. Due to the different preferences of DFRs for dihydroflavonols, these enzymes affect the biosynthesis of anthocyanin metabolites. Asn-type DFRs can use all three dihydroflavonols (DHK, DHQ, and DKM) as substrates, whereas Asp-type DFRs cannot use DHK efficiently [
8,
9,
10]. For example, as Asp-type DFRs from petunia and cymbidium cannot catalyze the conversion of DHK efficiently, they fail to produce brick red flowers in nature [
8,
9].
DFR is the key flavonoid biosynthetic enzyme contributing to the accumulation of pigments including anthocyanin and proanthocyanidin. In this study, we purified the two recombinant CmDFRs from bacterial expression (
Figure 5) and demonstrated their DFR activities (
Figure 6). During the process of protein purification, unlike the recombinant CmDFR-OB, the majority of recombinant CmDFR-RM protein was detected in the soluble fraction, which appeared to be associated with the C-terminal region of CmDFR-RM containing multiple hydrophilic residues (-SSSSKERT-). Although it cannot be ascertained whether these variations at the C-terminus of the two DFRs directly or indirectly affect the enzyme activity, at least the C-terminal hydrophilic region appears to affect the solubility of DFR protein.
CmDFR-OB and CmDFR-RM both contain the 134th Asn residue, which enable the catalysis of all three dihydroflavonol substrates including DHK. The result of in vitro assay showed that both enzymes produced leucopelargonidin and leucocyanidin from DHK and DHQ, respectively. Leucodelphinidin production was very scare or absent both of CmDFR-OB and CmDFR-RM, but it needs to expand its detection range to confirm. The enzymes both showed similar activities for leucocyanidin production. Interestingly, there was a striking difference in the production of leucopelargonidin. CmDFR-OB showed a dramatically reduced activity as compared with CmDFR-RM on leucopelargonidin production. This remarkable decrease in the activity of CmDFR-OB can be assumed to be due to the C-terminal truncation, suggesting that the variable C-terminus of DFR may be an important region conferring structural stability of binding pocket, especially near the B-ring binding site. A recent study showed that MaDFR harboring a point mutation at the 134th or 145th residue was still able to catalyze reactions using all three dihydroflavonol substrates [
17], which indicated that the 134th or 145th residue was not an absolute factor for substrate specificity of DFR. Therefore, it can be implied that the C-terminal variable region of DFR may be one of the important factors that determine substrate specificity of DFR.
Differences in the C-terminal region of DFR proteins have been associated with different substrate preferences and enzyme activities [
10,
18]. In poplar, two DFR proteins, PtrDFR1 and PtrDFR2, belonging to the Asp type DFRs, exhibited the variability at the C-terminus. The tobacco flower color change, due to anthocyanin accumulation, was observed in the ectopic expression of PtrDFR1, but not in that of PtrDFR2. In freesia, two DFR proteins (FeDFR1 and FeDFR2) classified into the same Asn type with high sequence similarity, but they exhibited the C-terminal variation each other. In vitro enzyme assay showed that FeDFR2 was able to convert DHQ into leucocyanidin, while FeDFR1 had no catalytic activity on DHQ. Taken together these results suggest that the variable C-terminus of DFR plays an important role in substrate preference and enzyme activity.
Several studies have indicated that anthocyanin biosynthesis takes place within a metabolon complex consisting of CHS, F3H, F3′H, DFR, and ANS, in which DFR might interact with ER-bounded cytochrome P450 to direct the metabolic flux towards anthocyanin biosynthesis [
19,
20]. In the metabolon complex, the F3′H and flavonoid 3′,5′-hydroxylase (F3′5′H) enzymes catalyze B ring hydroxylation. The sequential interaction between F3′H and DFR or between F3′5′H and DFR results in the accumulation of cyanidin- and delphinidin-derived anthocyanins, respectively. Additionally, substrate channeling between DFR and FLS for anthocyanin and flavonol biosynthesis contributes to color pattern formation in flowers [
4,
21].
Several reports have indicated that ray florets of chrysanthemum with bronze, pink, or purplish-red coloration accumulate only cyanidin derivatives, whereas white ray florets do not accumulate any anthocyanins [
14,
15,
22]. In the current study, we detected anthocyanins in the red-flowered cultivar RM but not in the white-flowered cultivar OB, indicating that anthocyanins were responsible for the coloration of these flowers. Additionally, we analyzed the expression of anthocyanin biosynthetic genes in ray floret of OB and RM to better understand anthocyanin biosynthetic mechanism in chrysanthemum (
Figure 7). Our study showed that most flavonoid biosynthetic genes, except for
CmCHS, were expressed at dramatically low levels in all floral developmental stage of OB as compared with that in RM. Interestingly, the transcript level of
CmF3H among EBG was hardly detected in OB during all flower developmental stages. Although all of LBG at FS1 in OB was expressed at a low level, it was not able to accumulate anthocyanin in the ray florets of OB. As well as, we verified that CmDFR-OB can catalyze the DHK and DHQ as substrates with in vitro assay (
Figure 6). Taken together these results suggest that the lack of
CmF3H transcripts may lead to the deficient of DHK and block off the metabolic flow toward anthocyanin biosynthesis in OB. Additionally, it is thought that the high activities of F3′H can fully convert DHK into DHQ, resulting in the predominant accumulation of B-ring dihydroxylated metabolites, which lead to accumulate cyanidin-based anthocyanins and quercetin derivatives [
14,
15]. On the basis of these results, we propose the anthocyanin biosynthetic pathway in ray florets of chrysanthemum, as shown in
Figure 9.
The
in planta assay confirmed that the ectopic expression of
CmDFR-RM restored the phenotype of Arabidopsis
tt3-1 to wild type, and therefore this mutant accumulated proanthocyanidin in its seeds and anthocyanin in its hypocotyls and mature leaves. However, the ectopic expression of
CmDFR-OB did not restore the accumulation of proanthocyanidin and anthocyanin to wild-type levels. In wild-type Arabidopsis seedling, cyanidin and pelargonidin accumulate at comparable levels and the major flavonol is kaempferol, [
23] indicating that Arabidopsis F3′H capacity only accepts a fraction of the total flavonoid metabolites. Therefore, the failure of
tt3-1 restoration by CmDFR-OB suggests that pelargonidin, which accounts for about half of anthocyanins, was barely produced in the transgenic lines due to dramatically decreased DHK acceptance of CmDFR-OB.
Taken together, our results indicate that the difference of DFR enzyme activity and lack of CmF3H transcripts may contribute to the anthocyanin accumulation of chrysanthemum. Further studies on the interactions between metabolon enzyme complex in chrysanthemum would provide additional insights into anthocyanin biosynthesis in the ray florets of this crop.
4. Materials and Methods
4.1. Plant Materials
Chrysanthemum plants were grown in a greenhouse under short day conditions at the National Institute of Horticultural and Herbal Science (Wanju, Korea). Two chrysanthemum cultivars named as OhBlang (OB) with white flower and RedMarble (RM) with red flower, respectively were used to analyze anthocyanin levels and gene expression. For the InDel marker analysis, we used twenty-two chrysanthemum cultivars. These cultivars are categorized based on their ray floret color as follows: white (BaekGang (BG), JimMa (JM), WonGyo 184 (WG184), BaekMa (BM), UnBaek (UB), BaekSeol (BS), JimMa2 (JM2), OB, SnowDream (SD), 13-62, AnGel (AG), 13-61, MoYa (MY), and PureAngel (PA)); pink (FreeMadona (FM); DonaPink (DP), CherryBlossom (CB), and PinkPride (PP)); and red (13-86, 12-71, BlackMarble (BM), and RM).
Transformation experiments were conducted using Arabidopsis thaliana transparent testa (tt) dfr mutant line tt3-1, which was obtained from the Arabidopsis Biological Resource Center (ABRC). All Arabidopsis plants were grown on 1/2 MS medium containing 1% sucrose or in soil under long-day conditions (LD, 16 h light/8 h dark) at 22 °C.
All samples were frozen rapidly in liquid nitrogen and kept at −80 °C. A portion of the samples was used for RNA extractions and anthocyanin measurements.
4.2. CmDFR Gene Cloning and Sequence Analysis
Genomic DNA was obtained from chrysanthemum leaves using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Total RNA was extracted from ray florets of flowers at different developmental stages from two different chrysanthemum cultivars OB and RM using Fruit-mate for RNA Purification solution (Takara, Otsu, Japan) and Plant RNA Purification Reagent (Invitrogen, Carlsbad, CA, USA), as described previously [
24]. The total RNA was purified using a FavorPrep™ Plant Total RNA Mini Kit (Favorgen, Changzhi, Taiwan), according to the manufacturer’s instructions. cDNA was synthesized from 2 μg of total RNA using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). The
CmDFR gene was amplified from cDNA and genomic DNA by PCR with PrimeSTAR
® HS DNA Polymerase (Takara) and the primer set CmDFR-F/R. All PCR fragments were subcloned into the pENTR/D-TOPO vector (Invitrogen) to validate the DNA sequences. All primer sequences are listed in
Supplementary Table S1.
4.3. InDel Analysis
Genomic DNA was extracted from the leaves of the 22 chrysanthemum cultivars using a DNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. A pair of PCR primers was designed to detect the 7-bp deletion in the sixth exon of
CmDFR (
Supplementary Figure S1). The PCR mixture contained 100 ng of genomic DNA, 5 pmol of each primer, 10 pmol of dNTPs, and 1 unit of PrimeSTAR HS DNA Polymerase in 1 × PrimeSTAR Buffer (Takara) in a total volume of 25 μL. The PCR conditions were as follows: An initial denaturation at 98 °C for 2 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, and an extension at 72 °C for 20 s, and a final incubation at 72 °C for 5 min. The PCR products were separated in a 5% nondenaturing polyacrylamide gel in 1 × TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0). Following electrophoresis, the DNA fragments were visualized by silver staining, as previously described [
26].
4.4. Expression and Purification of Recombinant CmDFR
The
CmDFR ORFs were amplified using primer sets designed as shown in
Supplementary Table S1. The PCR products were inserted into the pGEX-6P-1 vector linearized by
BamHI digestion using an In-Fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA, USA) in-frame with the sequence encoding the N-terminal glutathione
S-transferase (GST) tag. The resulting pGEX-6P-1:
CmDFR vectors were verified by sequencing and transformed into
Escherichia coli strain BL21 (DE3) cells (Novagen, Darmstadt, Germany). Transformed bacterial cells were cultured in 50 mL of LB broth containing 50 μg·mL
–1 ampicillin. Protein expression was induced by the addition of 0.1 mM isopropyl
β-
D-1-thiogalactopyranoside (IPTG) at 20 °C for 20 h. After centrifugation, the collected bacterial cells were lysed by sonication in 2 mL of sonication buffer containing 50 mM sodium phosphate (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride. Following centrifugation (13,000×
g, 4 °C, 10 min), 100 μL of Glutathione Sepharose 4B beads (GE Healthcare, Pittsburgh, PA, USA) were added to the soluble bacterial lysate and incubated at 4 °C for 2 h, with gentle rotation. The GST-tagged protein-bound beads were collected and washed five times with 1 × PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na
2HPO
4, and 2 mM K
2HPO
4 (pH 7.4)). For GST cleavage, a mixture of 4 μL of PreScission protease (GenScript, Piscataway, NJ) and 96 μL of cleavage buffer (50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT) was added to the beads and incubated for 3 h, at 4 °C. Following incubation, the beads were pelleted by centrifugation at 500×
g for 3 min and the eluate was transferred to a new tube. The beads were washed with two bed volume of cleavage buffer, and the washing solution was combined with the eluate. The concentrations of the GST-cleaved CmDFR proteins were equalized to 1.1 μg/μL by adding cleavage buffer, and the same volume of glycerol was added to obtain a final concentration of 0.55 μg/μL. These were used for assay and the rest were stored in −20 °C. The purified untagged proteins in the eluate were quantified by the Bradford method and verified by SDS-PAGE.
4.5. In Vitro Assay for CmDFR Activity
The enzymatic activities of the CmDFRs were assayed in a 200 μL reaction containing 3.3 μg of GST-cleaved CmDFR protein, 100 mM Tris-HCl (pH 7.5), 1 mM glucose 6-phosphate, 0.75 mM nicotinamide adenine dinucleotide phosphate reduced, 1 unit of glucose 6-phosphaste dehydrogenase, and 1 mM of dihydroflavonol substrates. The reactions were initiated by adding dihydroflavonol substrates and incubated at 30 °C for 50 min, and 10 μL acetic acid was added to stop the reaction. The reactants were extracted twice with 500 μL of ethyl acetate and dried using nitrogen gas. The products of the reactions were unstable leucoanthocyanidins; therefore, the residues were dissolved in 100 μL of acidic alcohol, n-butanol:HCl (95:5, v/v) to generate the corresponding stable anthocyanidins. Following incubation at 95 °C for 30 min, 10 μL of the solution was analyzed by high-performance liquid chromatography (HPLC).
4.6. HPLC Analysis
HPLC analysis was conducted on an LC-20A HPLC system with a diode array detector (Shimadzu, Kyoto, Japan). The separation of dihydroflavonol substrates and anthocyanidin products was accomplished on an Inertsil-ODS3 C18 column (5 μm, 250 × 4.6 mm, GL Science). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient profile was optimized as follows: 0 min, 95% A/5% B; 30 min, 45% A/55% B; 45 min, 35% A/65% B; 50 min, 0% A/100% B; 52 min, 95% A/5% B; and 60 min, 95% A/5% B. The flow rate was 1 mL·min−1, and the column temperature was maintained at 30 °C. The detection wavelength was 288 nm for dihydroflavonols and 520 nm for anthocyanidins.
4.7. Chemical Standards
(±)-DHK, (±)-DHQ, and (±)-DHM were purchased from Sigma-Aldrich (St. Louis, MO, USA), and pelargonidin chloride, cyanidin chloride, and delphinidin chloride were purchased from Extrasynthese (Extrasynthese, Genay Cedex, France). The dihydroflavonols were prepared as 100 mM stock solutions in DMSO, and the anthocyanidins were prepared as a 100 mM solution in 50% methanol containing 1.2 N HCl.
4.8. Measurement of Total Anthocyanin Contents
Total anthocyanin contents were measured from ray florets of flowers at the following different developmental stages: FS1, 6.0 weeks under short-day (SD) treatment, on which the first coloration of ray florets was first visible; FS2, 7.0 week under SD treatment that is the date of early flowering; and FS3, 7.5 weeks under SD treatment that is the date of full flowering. As described by [
24], powdered ray floret samples were incubated in 600 μL extraction buffer (methanol containing 1% HCl) for 6 h, at 4 °C, with moderate shaking. Following the addition of 200 μL water and 200 μL chloroform, the samples were centrifuged at 14,000×
g for 5 min at 4 °C to sediment the plant material. The absorbance of the supernatant was recorded at 530 nm (A
530) and 657 nm (A
657) using a microplate reader. Anthocyanin content was determined using the following equation: A
530 − 0.33 × A
657. The anthocyanin content in each sample was measured in three independent experiments.
4.9. Quantitative Reverse-Transcription PCR (qPCR)
Total RNA was prepared from ray florets, as described above. cDNA was synthesized from 2 μg of total RNA using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT).
qPCR was performed using AccuPower 2x Greenstar qPCR Master Mix (Bioneer, Daejun, Korea) and a Bio-Rad CFX96 Detection System (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. Gene expression was normalized using the elongation factor 1α (
EF1α) as the reference gene. The gene-specific primers used for qPCR analysis are listed in
Supplementary Table S1. Three biological replicates were performed per sample.
4.10. In Planta Assay of CmDFR Function
The plasmid used for stable transformation of Arabidopsis was constructed as follows: The ORFs of CmDFR isolated from cultivars OB (CmDFR-OB) and RM (CmDFR-RM) were subcloned into the pENTR/D-TOPO vector (Invitrogen) and incorporated into the Gateway destination vector pB7WG2D (VIB-Ghent University, Ghent, Belgium) via several Gateway cloning steps. The resulting vector was maintained in Agrobacterium tumefaciens strain GV3101 and transformed into the Arabidopsis tt3-1 mutant using the floral dip method. Transformed Arabidopsis seeds were grown in soil under LD conditions at 22 °C. Transgenic Arabidopsis plants were selected by spraying the plants with 0.3% basta solution. Homozygous T2 lines were subjected to evaluate expression level of exogenous CmDFR gene and phenotypic investigation. Arabidopsis elongation factor 1α (EF1α) gene was used as internal reference.
4.11. Statistical Analysis
For qPCR analysis, results were represented as mean values ± SD from three biological replicates. For the analysis of anthocyanin contents, experimental data were presented as mean values ± SD of three biological replicates. Statistical significance was determined by one-way ANOVA followed by a Duncan’s multiple range tests.