Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars

Anthocyanins are secondary metabolites in land plants that contribute to the colors of leaves and flowers, and are nutritionally valuable components of the human diet. The DFR gene plays an important role in the anthocyanin biosynthetic pathway. In this study, we investigated the regulation of DFR expression and in different Malus crabapple cultivars that show distinct patterns of leaf coloration, and how it influences leaf anthocyanin accumulation and coloration. Specifically, we studied the ever-red leaved cultivar ‘Royalty’, the ever-green leaved cultivar ‘Flame’ and the spring-red leaved cultivar ‘Radiant’. RT-PCR analysis showed that the expression of McDFR1 correlated with the expression of a MYB transcription factor, McMYB10, and with anthocyanin accumulation. We isolated five McDFR1 promoter fragments from the three cultivars and identified four different fragments (F1–4) that were present either in several cultivars, or only in one. Yeast one-hybrid and electrophoretic mobility shift assay analyses showed that McMYB10 could bind to all the McDFR1 promoters, except McDFR1-Ra2. The F1, F2 and F3 fragments did not affect McMYB10 binding to the McDFR1 promoters; however, we found evidence that the F4 fragment suppressed binding, and that the MYBGAHV amino-acid sequence maybe an important cis-element for McMYB10 protein binding. This information has potential value for strategies to modify plant color through genetic transformation.


INTRODUCTION
Anthocyanins comprise a class of water-soluble flavonoids pigments in plants that contribute to the color of flowers, fruit, stems and leaves. 1,2 They also function in vegetative tissues that provide protection against UV and high light irradiation, as antioxidants to scavenge reactive oxygen species (ROS), and as antimicrobial agents during defense responses. [3][4][5][6] Anthocyanins are also potentially beneficial components of the human diet, 6,7 and they can act as antioxidants, are anti-carcinogenic, 8,9 antiinflammatory 10 and may help support both diabetes prevention and treatment, 11 and heart health. 12 Over the past few decades, the anthocyanin biosynthetic pathway has been well characterized in plants, such as arabidopsis (Arabidopsis thaliana), petunia (Petunia hybrida), maize (Zea mays), snapdragon (Antirrhinum majus) and apple (Malus domestica). [13][14][15][16][17] Most of the genes encoding enzymes responsible for each step in the flavonoid biosynthetic pathway have been identified, 18 and it has been established that dihydroflavonol 4-reductase (DFR) plays a key role in the formation of common and condensed anthocyanins ( Figure 1). 18 DFR is one of the rate-limiting enzymes that catalyzes the production of flavan-3,4-diols (leucoanthocyanidins) via the reduction of three colorless dihydroflavonols: dihydrokaempferol (DHK), dihydroquercetin (DHQ); and dihydromyricetin (DHM). These three compounds are also intermediates in flavonol biosynthesis through the flavonol synthase reaction, using NADPH as a cofactor. 19,20 A number of DFR genes have been characterized from a wide range of plant species, including Camellia sinensis (tea), 21 Medicago truncatula (Medicago), 22 Ipomoea batatas Lam (sweet potato), 23 Ginkgo biloba (ginkgo) 24 and Populus trichocarpa. 25 It has also been shown that overexpression of different DFR genes in tobacco (Nicotiana benthamiana) flowers promotes anthocyanin biosynthesis, corresponding to an increase in red pigmentation. 26 In addition, suppressing IbDFR in sweet potato led to a decrease in anthocyanin accumulation and reduced the tolerance to abiotic stress. 23 DFR not only regulates levels of anthocyanin, but also shows substrate specificity, resulting in the accumulation of different types of anthocyanins. For example, petunia (Petunia hybrida (J. D. Hooker) Vilmorin) and cymbidium (Cymbidium faberi Rolfe) lack varieties with brick red/orange colored flowers due to a lack of pelargonidin-based anthocyanins because DFRs do not utilize dihydrokaempferol as a substrate. 27,28 Furthermore, overexpressing MtDFR in rice also changed several metabolites except anthocyanins, including the concentrations of amino acids, sugars and metals. 29 30 In addition, overexpression of RrDFR1 (Rosa rugosa) and PhDFR1 (Petunia hybrida) genes in tobacco displayed downregulation of the endogenous NtFLS gene, and the promotion of anthocyanin synthesis. The relative expression levels of NtCHS and NtFLS were significantly downregulated and NtANS genes was upregulated in DFR transgenic lines. 26 In plants, MYB (v-myb avian myeloblastosis viral oncogene homolog), bHLH (basic-helix-loop-helix) and WD40 (Trp- Asp 40) transcription factors (TFs) often form MYB-bHLH-WD40 (MBW) complexes and play important roles in secondary metabolism, development, signal transduction and disease resistance. It has been shown that several MYB TFs are involved in positively regulating the expression of DFR. 31 In A. thaliana, the expression of the MYB75/PAP1 gene is induced by light and precedes the expression of both MYB90/PAP2 and several structural genes (CHS, DFR, F3H and LDOX). 32 Moreover, the apple TFs MdMYB10 and MdMYB1 control the red pigmentation in the fruit flesh and skin, respectively, 33 and the corresponding proteins, MdMYB10 and MdMYB1, can form complexes with bHLH genes to trans-activate the DFR promoter and promote anthocyanin accumulation. 34 miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes DFR through destabilization of a MYB-bHLH-WD40 (PAP1, TT8 and TTG1) transcriptional activation complex. 32 In contrast, several MYB transcription factors also have been proved that they can suppress the expression of DFR. AtMYBL2 interacts with TT8 (TRANSPARENT TESTA 8) to reduce anthocyanin biosynthesis by suppressing the expression of DFR. 33 The expression of the AtMYB7 gene is induced by salt stress, and represses several flavonoid pathway genes, including DFR and UGT. 35 Furthermore, MYBCORE, MYBGAHV cis-regulatory elements have been proved that involved in MYB transcription factors binding to several promoters to regulate gene expression, such as MYB101 regulates fertilization in Arabidopsis thaliana by binding to the MYBGAHV elements in the promoters of downstream genes, and MYB5 and TT2 regulates proanthocyanins accumulation in Arabidopsis seed coat by binding to the MYBCORE elements in the promoter of DFR. 36,37 Structural differences in promoter regions contribute to the difference in expression of the target genes regulated by TFs. 38 In apple, a TATA-box insertion in the IRT1 promoter is responsible for increasing Fe uptake, and its presence correlates with an increase in transcriptional activation by specific binding of the TF, IID. 39 Meanwhile, promoter structure also affect anthocyanins-related gene expression in plants. Subsequent analysis showed that a rearrangement of the 23-bp sequences in the promoter regions of MdMYB10 and McMYB10 was responsible for the difference in gene expression between the white-and red-fleshed apples and red-leaf color crabapples. 33 Specifically, the R1: MdMYB10 (McMYB10) promoter has a single MdMYB10 (McMYB10) binding motif, and is only present in white-fleshed apples, while the R6: MdMYB10 (McMYB10) promoter, which is present in red-fleshed apples, has five additional tandem repeats of the MdMYB10 (McMYB10) binding motif. Our studies in Malus crabapple have shown that a 743-bp fragment missing of McCHS promoter was found in ever-green leaf color crabapple cultivar, and the missing sequence contained two MYBPLANT elements and three MYB-CORE elements that are essential for MYB transcription factor binding and inducing anthocyanin biosynthesis. So, we conclude that different structures in the chalcone synthase (McCHS) promoter affect the binding of MYB TFs and the expression of McCHS, and lead to the accumulation of different anthocyanins in crabapple cultivars with different leaf colors. 40 However, little is known about the basis for the differences in DFR expression caused by MYB TFs.
Malus crabapples are collectively an economically important germplasm resource for ornamental plants, providing numerous landscape species. They also exhibit stress resistance and provide valuable research material to investigate the mechanisms of anthocyanin accumulation and color formation, reflected by the diversity of leaf, flower and fruit coloration. 41 In this current study, the expression analysis showed that McDFR1 may play an important role in anthocyanins biosynthesis in crabapple leaves, hence we examined differences in the McDFR1 promoter sequences and functions in three different types of Malus crabapple: Malus cv. 'Flame', Malus cv. 'Royalty' and Malus cv. 'Radiant'. We investigated whether variations in the DFR1 promoter may affect the expression of DFR1 in different colored leaf crabapple cultivars and provide evidence that the McDFR1 promoter has potential applications to improve plant color via genetic transformation approaches.

Material and growth conditions
Three crabapple cultivars were used in this study: 'Royalty', an ever-red leaved cultivar; 'Flame', an ever-green leaved cultivar; and 'Radiant', which has red young leaves that turn green as they mature. Eight-year-old crabapple trees grafted onto a Malus 'Balenghaitang' stock were grown in the Crabapple Germplasm Resources Nursery, Beijing University of Agriculture (40.l″ north latitude, 116.6″ longitude). In mid-late April (18-22°C) and on sunny days, we selected six trees of each cultivar that showed similar growth and collected leaf samples from annual branches growing in all four compass directions on the outer edge of each canopy. Leaves of 'Royalty', 'Radiant' and 'Flame' were collected at 10 developmental stages (3,6,9,12,15,18,21,24,27 and 30 days after budding). Branches to be used for the experiments were marked before budding. All samples were immediately frozen in liquid nitrogen and stored at − 80°C.

Cloning and analysis of the McDFR1 promoters
To analyze the differences in McDFR1 promoter sequences of the three different leaf colored crabapple cultivars, genomic DNA was isolated from 'Royalty', 'Radiant' and 'Flame' leaves using the Plant Genomic DNA Kit  Error bars indicate the s.e.m. ± s.e. of three replicate measurements. Different letters above the bars indicate significantly different values (P o0.05) calculated using one-way analysis of variance (ANOVA) followed by a Tukey's multiple range test. ND, no detection. 1-10 represents stage 1-10 of leaf development stages. 1, 3 days after budding; 2, 6 days after budding; 3, 9 days after budding; 4, 12 days after budding; 5, 15 days after budding; 6, 18 days after budding; 7, 24 days after budding; 8, 21 days after budding; 9, 27 days after budding; 10, 30 days after budding.
(TIANGEN BIOTECH CO., LTD, Beijing, China). The 5′-upstream sequences were amplified by hi-TAIL PCR 44 the primers are shown in Supplementary  Table S1. The primers for hi-TAIL PCR (high-efficient thermal asymmetric interlaced PCR) were designed based on the McDFRs cDNA sequence (GenBank Accession: FJ817487, AF117268). The upstream-flanking sequence of McDFR1 was isolated using hi-TAIL PCR. The hi-TAIL PCR is a method to isolate upstream (promoters) and downstream sequences of the known coding sequences. Long (33-34 nucleotides) arbitrary degenerate (LAD) primer with a higher degree (2304 or 6912 folds) of degeneracy is used to create primer-binding sites efficiently along the unknown target sequences and used for the first amplification. An additional sequence (AC1, 18 nucleotides with T m = 58°C, used for the second and third amplification) identical to the 5′ part of the LAD primer is tagged to the 5′ end of a long primer (T m 468°C) specific to the known sequence. The forward special primers (McDFR1-promoter1, McDFR1-promoter2, and McDFR1-promoter3; Supplementary Table S1) used in three reactions were based on the McDFR1 coding sequence (GenBank Accession: FJ817487). All PCR products were sub-cloned into the pGEM T-Easy Vector (Promega, Madison, WI, USA) and transformed into Escherichia coli DH5α cells and sequenced. The cis-elements were analyzed using the PLACE database (http://www.dna.affrc.go.jp/PLACE/).

RNA extraction and semi-quantitative RT-PCR
Total RNA was extracted from crabapple leaves using the RNA Extract kit (Aidlab, Beijing, China) according to the manufacturer's instructions. DNase I (TaKara, Ohtsu, Japan) was added to remove genomic DNA, and the 1 μg RNA samples were subjected to cDNA synthesis using the Access RT-PCR System (Promega), according to the manufacturer's instructions.
Semi-quantitative RT-PCR was carried out in 20 μL reactions with 2 μL of 10 × diluted cDNA template, and McActin was used as the internal control. 45 transcription system (Promega) PCR amplification system with the following conditions: initial denaturation at 95°C for 3 min, followed by  Table S1. 46 The expression levels of McDFR1, McDFR2 and McMYB10 in crabapple were analyzed using qRT-PCR and the SYBR Green qPCR Mix (TaKaRa) and the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA), according to the manufacturers' instructions. The PCR primers were designed using NCBI Primer BLAST and are listed in Supplementary Table  S1. qRT-PCR analysis was carried out in a total volume of 20 μL containing 9 μL of 2 × SYBR Green qPCR Mix (TaKaRa), 0.1 μM specific primers (each), and 100 ng of template cDNA. The reaction mixtures were heated to 95°C for 30 s, followed by 39 cycles at 95°C for 10 s, 50-59°C for 15 s and 72°C for 30 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. The transcript levels were normalized using the Malus 18S ribosomal RNA gene (DQ341382, for apple and crabapple) as the internal controls and calculated using the 2 (− ΔΔCt) analysis method. 46 Yeast one-hybrid assay A yeast one-hybrid system was used to assay the relationship between the McMYB10 protein and the McDFR1 promoters from the three Malus

McDFR expression and anthocyanin accumulation during leaf development
McDFR is known to play an important role in anthocyanin biosynthesis. 49 There are two DFR homologs in crabapple, and the full-length McDFR cDNAs of these two genes were cloned from cDNA libraries that from the leaf of ever-red leaf color crabapple cultivar 'Royalty', and named We observed that in the ever-red 'Royalty' cultivar, the abundance of anthocyanins was relatively high in the first six developmental stages, before decreasing to low levels in the last four stages. Anthocyanin levels increased during the first five development stages and a gradual decrease was observed in the last five development stages in the spring-red crabapple cultivar   Table S3).
Taken together, these data suggest that  (Table 1). However, the number and the positions of MYB1AT, MYB1LEPR, MYBGAHV and MYBST1 varied. MYB1AT and MYBST1 were located 800 bp upstream of the ATG transcriptional start site and were present in various numbers, while MYB1LEPR were present 600 bp upstream from the ATG in the two promoters that did not contain

DISCUSSION
There is considerable interest in the breeding of ornamental plants with colored leaves, especially those with red/purple leaves, which is mainly due to the accumulation of anthocyanins. [52][53][54] However, the molecular mechanism of red color formation in leaves is still unclear. DFR is a key enzyme in the catalysis of the stereo-specific reduction of dihydroflavonols to leucoanthocyanidins, which uses NADPH as a cofactor and is located in a key branch point in the anthocyanin biosynthetic pathway. 55 The expression level variations of DFR determine the color of leaves, flowers and fruits in many plants, [56][57][58] and given the central role of DFR in anthocyanin accumulation, we investigated the possible regulatory mechanism by which MYB TFs control DFR gene expression in crabapple leaves.
HPLC analysis and transcript quantification suggested that red leaves were associated with higher anthocyanin accumulation, which was consistent with increased transcript levels of McDFR and McMYB10 (Figure 2). Several studies have shown that MYB10 can activate the expression of DFR and promote anthocyanin accumulation. In Arabidopsis thaliana, AtDFR is a target gene of MYB75/PAP1 and its expression can be enhanced by an elevated expression of MYB75. 59 MdMYB10 and MdMYB1 have been shown to be able to bind the DFR promoter and promote anthocyanin accumulation in apple skin. 34 Therefore, we hypothesized that  (Figure 5a) was detectable, we hypothesize that there was no anthocyanin accumulation in the green leaves as a consequence of low MYB10 expression, and the low level of expression of genes in the anthocyanin biosynthetic pathway.
In a previous study, 1 kb insertion in the promoter that increases the expression of citrate transporter gene HvAACT1 in several Al-tolerant barley cultivars. 60 In addition, a deletion in the promoter of a mitochondrial molybdenum transporter gene (MOT1) in Arabidopsis is associated with reduced gene expression and low molybdenum (Mo) levels in the shoot. 61 The same nucleotide polymorphisms were also appeared in anthocyaninsrelated genes. An extra fragment (255 bp) in the promoter of TfF3′ H1 in reddish flower tulip sport Tulipa fosteriana hampered the accumulation of cyanidin anthocyanins through the reduction of TfF3′H1 transcription. 62 A 23 bp repeat motif in the upstream regulatory region of MYB10 alleles was found only in red-fleshed apples and red leaved crabapples. 33,63 Moreover, this promoter allele was shown to be responsible for the increased accumulation of anthocyanins, and the number of repeat units correlated with an increase in trans-activation by the MYB10 protein. 33 (Figure 5b), and we also found that the MYBGAHV element that was not present in McDFR1-Ra2 and McDFR1-Ra2 (without F4) affected McMYB10 binding to the DFR1 promoter to increase DFR expression. We concluded that the F4 fragment in the McDFR1-Ra2 promoter sequence determined whether or not MYB10 binds to the McDFR1 promoter, and that the MYBGAHV element may be an important cis-element in regulating DFR1 expression by MYB10. Meanwhile, we also analyzed the potential bHLH or WD40 binding sites in F4 fragment, the results showed that F4 fragment lack related transcription factors binding sites. Hence, we speculate that McDFR1-Ra1 promoter was preferentially used in 'Radiant' to make the leaf present red color in early development stages. And the McDFR1-Ra2 promoter was keep low activity in 'Radiant' during leaf development.
PCR analysis showed that both the R6 and R1 promoters of McMYB10 were present in the genome simultaneously in springred crabapple cultivars, 33 and the same phenomenon (different promoters of one gene exist in one cultivars) was observed in the McDFR1 promoter in spring-red crabapple cultivars. The McDFR1-Ra2 promoter, which contains the important repressive F4 fragment and the McDFR1-Ra1 promoter, which is strongly bound by McMYB10, are both present in the spring-red crabapple cultivar 'Radiant'. We therefore deduce that the key anthocyanin-related genes contain activated and non-activated promoters simultaneously, and this may be a characteristic of the genomes of cultivars where red and green leaf color co-exist.
Alteration of the expression of DFR in plants represents an excellent strategy to modify the content of anthocyanins and flavonoids. In this regard, a suitable promoter is critical for the expression of exogenous genes, and our data suggest that the F4 fragment and the MYBGAHV cis-element may be used as valuable tools for the regulation of gene expression.