While there is a range of colors found in plants, the predominant color is green. Pigments in plants have several roles, e.g., photosynthesis, signaling, defense or heat exchange. In order to stand out from the predominant green colors of leaves and stems, plants have flowers (and fruits) with many colors and sometimes multiple color patterns (Miller et al., 2011), which are evloved to attract pollinators as visual signals. Floral pigments mainly include carotenoids, betalains, and flavonoids (Tanaka et al., 2008). In most plant species, flower coloration is primarily caused by flavonoids, and the flavonoid family encompasses at least 6000 molecules, chiefly divided into phlobaphenes, aurones, isoflavonoids, flavones, flavonols, flavanols, and anthocyanins (Hichri et al., 2011). Among them, anthocyanins are the most common pigments found in flowers and fruits (Tanaka et al., 2008; Morita et al., 2014) and, thus, are of particular importance.

Anthocyanins are a major class of flavonoids showing bright coloration ranging from blue to orange. Actually, two different types of anthocyanins, 3-hydroxyanthocyanins and 3-deoxyanthocyanins, can be formed in plants (Styles and Ceska, 1975; Halbwirth et al., 2003; Kawahigashi et al., 2016). In contrast to the rare 3-deoxyanthocyanins which have been found only in a few plant species, the ubiquitous 3-hydroxyanthocyanins distribute widely in nature. Thus, the common anthocyanins usually refer to the widely existing 3-hydroxyanthocyanins. Over the past few decades, the biosynthetic pathway of anthocyanins has been well established in plants. They are derived from phenylalanine via the general phenylpropanoid pathway (Figure 1). Actually, the general phenylpropanoid pathway also provides precursors for several branches leading to thousands of compounds, for example, lignins could be synthesized from ρ-coumaroyl-CoA by several enzymes containing cinnamoyl-CoA reductase (CCR).

Typically, two classes of genes are confirmed to be involved in the flavonoid pathway: structural genes encoding enzymes that directly participate in the formation of flavonoids, and regulatory genes that control the expression of the structural genes. As shown in Figure 1, one molecule of ρ-coumaroyl-CoA and three molecules of malonyl-CoA are catalyzed by chalcone synthase (CHS) to generate naringenin chalcone, which is isomerized to naringenin by chalcone isomerase (CHI). Then, flavanone 3-hydroxylase (F3H) catalyzes the hydroxylation of naringenin to produce dihydrokaempferol (DHK, one hydroxyl group), which is further hydroxylated at the B-ring to form dihydroquercetin (DHQ, two hydroxyl groups) and dihydromyricetin (DHM, 3 hydroxyl groups) by F3′H and F3′5′H, respectively. These genes are usually regarded as early biosynthetic genes (EBGs) in flavonoid biosynthetic pathway. Subsequently, dihydroflavonol 4-reductase (DFR) catalyzes dihydroflavonols to leucoanthocyanidins, which are then converted to stable anthocyanins by leucoanthocyanidin di-oxygenase (LDOX) and flavonoid 3-O-glucosyltransferase (3GT) (Holton and Cornish, 1995; Forkmann and Martens, 2001; Jaakola, 2013). And these genes are designated as late biosynthetic genes (LBGs). Comparably, the 3-deoxyanthocyanidin synthesis pathway shared the same early steps, which are consecutively catalyzed by CHS and CHI. Then the naringenin can be converted to apiforol by flavanone 4-reductase (FNR) or eriodictyol by F3′H. Subsequently, eriodictyol can also be catalyzed into luteoforol by FNR (Lo and Nicholson, 1998; Shih et al., 2006; Liu et al., 2010). In addition, other flavonoids can also be synthesized by partially overlapping or competing pathways. For example, the substrate dihydroflavonol of DFR can be catalyzed by flavonol synthase (FLS) to produce flavonols, and leucoanthocyanidins that result from DFR can be converted to proanthocyanidin by leucoanthocyanidin reductase (LAR), while anthocyanidins resulting from LDOX can also be converted to another kind of proanthocyanidin by anthocyanidin reductase (ANR) (Davies et al., 2003; Xie et al., 2003; Xie and Dixon, 2005; Martens et al., 2010; Yoshida et al., 2010). Herein, FNR, DFR, CCR, ANR, and LAR are important components of the NADPH-dependent reductase superfamily, which fulfill versatile roles in the biosynthesis of plant secondary metabolites.

Dihydroflavonol-4-reductase is a pivotal oxidoreductase (EC 1.1.1.219) catalyzing the NADPH dependent stereospecific reduction of dihydroflavonols, e.g., dihydrokaempferol, dihydroquercetin and dihydromyricetin, to generate leucopelargonidin (LEUP), leucocyanidin (LEUC), and leucomyricetin (LEUM), respectively (Halbwirth et al., 2006; Petit et al., 2007). The substrate specificity of DFR results in different kinds of anthocyanins, mainly delphinidin derivatives, cyanidin derivatives and pelargonidin derivatives. Mutations in DFR may explain the color transition in some species such as Andean genus Iochroma (Des Marais and Rausher, 2008; Smith and Rausher, 2011; Smith et al., 2013). To our knowledge, various DFR genes have so far been isolated from a wide range of plant species, such as Lotus japonicus (Shimada et al., 2005), Camellia sinensis (Singh et al., 2009), Medicago truncatula (Xie et al., 2004), Malus domestica (Fischer et al., 2003), Pyrus communis (Fischer et al., 2003), Citrus sinensis (Piero et al., 2006), Ipomoea batatas Lam (Wang et al., 2013), Ginkgo biloba (Cheng et al., 2013), and Populus trichocarpa (Huang et al., 2012). However, most of the genes were isolated from dicot plants and few studies have focused on the functionally divergence of the members in the DFR gene family, especially in monocot species.

In addition to the structural genes aforementioned, the regulatory mechanism involved in the flavonoid biosynthesis has also been well characterized in plants. Three distinct transcription factor (TF) gene families, containing R2R3 -MYB, basic helix -loop -helix (bHLH) and WD40 repeats (WDRs), comprise a regulatory protein complex (designated as MBW complex) regulating multiple flavonoid metabolisms. At least six MYBs, i.e., PAP1, PAP2, MYB113, MYB114, TT2, MYB5, and three bHLHs, i.e., TT8, GL3, EGL3, and one WD40 (TTG1) regulating the DFR expression in Arabidopsis thaliana have been well elucidated (Baudry et al., 2004; Gonzalez et al., 2008; Pires and Dolan, 2009; Petroni and Tonelli, 2011; Patra et al., 2013; Xu et al., 2014, 2015). To date, some common TF components of the MBW complex regulating DFR expression have been found in maize, petunia, tobacco, and other angiosperms, especially in dicots (Xue et al., 2011; Jaakola, 2013; Xu et al., 2015).

Freesia, a monocotyledonous genus of herbaceous perennial flowering plants in the family Iridaceae, is native to the eastern side of southern Africa, and then widely distributes in the world as a cut flower. The freesia flower colors available include red, pink, yellow, white, blue, lavender, purple, and various bicolors. As a result, it has the potential to be a model system for investigating of flavonoid biosynthesis in monocots, particularly for the flower pigmentation. Our previous studies have confirmed the composition of anthocyanin aglycons, i.e., delphinidin, petunidin, malvinidin, peonidin, and cyanidin, and flavonols, i.e., kaempferol and quercetin derivatives, in Freesia hybrida “Red River^®,” Furthermore, we also found that the accumulation profile for anthocyanins was the opposite of that for flavonols during the flower development process (Sun et al., 2016). In addition, proanthocyanidins were detected (Li et al., 2016) which also showed special accumulation patterns. Therefore, the complicated flavonoid compounds in freesia flowers indicated a sophisticated biosynthetic pathway and transcription regulation network for the flavonoid accumulation. So far, two anthocyanin biosynthetic genes, Fh3GT1 and FhCHS1, as well as two bHLH regulatory genes, FhGL3L and FhTT8L, were isolated and functionally verified (Sui et al., 2011; Sun et al., 2015, 2016; Li et al., 2016). However, no DFR-like genes have been isolated and functionally characterized, which is worthy of further concerns because their particular positions in flavonoid biosynthetic pathway.

In the present study, eight putative DFR-like genes were firstly cloned from flowers of a universal cultivar of F. hybrida, “Red River^®,” and only three of them were phylogenetically clustered into the DFR subgroup, designated as FhDFR1, FhDFR2, and FhDFR3, respectively, which were further functionally characterized. Their temporal and spatial expression profiles were detected and potential roles in planta were investigated through introducing into Arabidopsis dfr (tt3-1) mutant plants. Furthermore, biochemical properties of FhDFR proteins were also determined. Results indicated that dihydroflavonol 4-reductases performed the crucial roles in the anthocyanin biosynthesis because of substrate specificities, and their functions were at least partially divergent. As expected, the three FhDFR genes might be controlled by the common MBW complex with diverse regulation efficiencies, because Arabidopsis leaf protoplasts transient expression analysis demonstrated that the earlier characterized FhGL3L and FhTT8L could regulate the expression of FhDFR1/FhDFR2 and FhDFR3 coupled with Arabidopsis endogenous MYB-type TF, AtPAP1, and the promoter of FhDFR3 was activated more extensively. Based on the results aforementioned, a model that elucidated the anthocyanin biosynthesis in F. hybrida was proposed. To our knowledge, this is the first report of the identification of dihydroflavonol 4-reductase gene family in F. hybrida, and the results will not only provide new insights into the flavonoid biosynthesis in monocot plants but also contribute to the study of evolution of DFR gene families in angiosperms.

Dihydroflavonol-4-reductase (DFR) genes presented strong sequence conservation in plants. In flavonoid biosynthesis, DFR is a member of the short chain dehydrogenase/reductase (SDR) superfamily which contained a highly conserved NADP-binding domain (Johnson et al., 1999, 2001; Martens et al., 2002). In this study, we firstly cloned eight putative homologous cDNA sequences of NADPH-dependent reductase genes in F. hybrida. Interestingly, FhDFR1, FhDFR2, and FhDFR3 tended to be DFR-like proteins, whereas other five proteins were more closely related to CCRs through the manual BLASTX search (Supplementary Table S1). Furthermore, FhDFR1 and FhDFR2 showed high similarities in both nucleotide and amino acid sequences. cDNAs synthesized from total RNA of several tissues were used to evaluate the ratio between FhDFR1 and FhDFR2. Primer sets binding to the same site of FhDFR1 and FhDFR2 were used and at least 30 clones were sequenced. Results showed the ratio between FhDFR1 and FhDFR2 was about 1:1. Bioinformatic analysis found that the proteins encoded by these two genes as well as FhDFR3 had typical functional domains of DFR proteins, including a conserved NADPH binding motif “VTGAAGFIGSWLIMRLLERGY,” a substrate specificity selective domain and several specific loci of the conservative short-chain dehydrogenase/reductase family (Johnson et al., 1999; Martens et al., 2002). In order to further investigate the potential functions of DFR-like genes in F. hybrida, we generated a phylogenic tree containing the other characterized NADP-dependent reductases such as DFR, CCR, ANR, LAR, and FNR proteins in other species. As shown in Figure 2, both the amino acid alignment and phylogenic tree revealed a higher similarity between FhDFR1, FhDFR2, FhDFR3 and other plants originated and characterized DFRs (Figure 2), indicating their potential “DFR-like” catalytic properties. Furthermore, FhDFR1, FhDFR2, and FhDFR3 phylogenetically clustered into the same subgroup with DFRs from other monocot plants. Thus, it can be deduced that the divergence of DFRs most likely occurred after the division of monocots and dicots, and that there might be homologous DFR genes in different plants (Liew et al., 1998).

Previous studies demonstrated that substrate specificity of DFR could be influenced by a presumed substrate-binding region composed of 26 amino acid residues, especially the single amino acid residue at about residue 134 (Johnson et al., 2001). Consequently, DFRs could be divided into three types according to differences at this position, i.e., Asn-type DFRs, Asp-type DFRs, and non-Asn/Asp-type DFRs, the amino acid residue of the 134 position was asparagine residue (Asn), aspartic acid (Asp) and neither Asn nor Asp, respectively (Johnson et al., 2001). Generally, Asn-type DFRs could utilize all the three dihydroflavonols, DHK, DHQ, and DHM, as substrates, while Asp-type DFRs could not catalyze DHK efficiently (Forkmann and Ruhnau, 1987; Helariutta et al., 1993; Tanaka et al., 1995; Johnson et al., 1999). However, FhDFR1 and FhDFR2 belonged to Asn-type DFRs, no pelargonidin was detected in flowers of F. hybrida, and no LEUP was detected in the in vitro catalytic activity assays, which was consistent with ApDFR1 in Agapanthus praecox ssp.orientalis (Leighton) (Mori et al., 2014). Commonly, Asn-type DFRs are widely distributed in plants, whereas Asp-type DFRs are found in limited plant species that are scattered throughout the eudicots (Shimada et al., 2005). In contrast, FhDFR3, as well as IhDFR from Iris × hollandica, broke the rule, which belonged to monocotyledonous Asp-type DFRs.

The expression patterns of FhDFR genes of F. hybrida were tested temporally and spatially. In the development process of flowers, the amount of anthocyanin increased gradually and peaked when flowers fully opened, whereas the proanthocyanidins were constitutively accumulated at a relative low level (Li et al., 2016). FhDFR1/FhDFR2 was parallel well to the anthocyanin accumulation as well as FhDFR3, which showed a high expression level at the late stage of the flower development process. In addition, both anthocyanin and proanthocyanidin accumulated more extensively in flower tissues than vegetative tissues, and petal and torus were the dominant tissues for anthocyanin and proanthocyanidin biosynthesis, respectively (Li et al., 2016), which was also synchronous to expression patterns of FhDFR1/FhDFR2 and FhDFR3 in these tissues. However, the expression level of FhDFR3 was significantly higher than FhDFR1/FhDFR2 genes in all tested tissues, which might imply their partial or complete redundant roles. The spatial and temporal expression characteristics of FhDFR genes were found similar in several other species (Nakatsuka et al., 2003; Li et al., 2012; Mori et al., 2014).

In order to investigate the functional divergence of the three FhDFR genes in the flavonoid biosynthesis, FhDFR1, FhDFR2, and FhDFR3 were introduced into Arabidopsis tt3-1 mutants, and the results showed that cyanidin derivatives could be detected in the transgenic plants overexpressing exogenous FhDFR1, FhDFR2, FhDFR3 genes, indicating that these three genes could utilize DHQ as substrate in Arabidopsis. Because the contents of the cyanidin derivatives were significantly lower compared with the wild type plants, it can be deduced that FhDFR1, FhDFR2, FhDFR3 might have relative weaker catalytic efficiency for DHQ in contrast to AtDFR itself. On the other hand, no pelargonidin derivatives were detected in transgenic plants, implying that FhDFR1, FhDFR2, FhDFR3 might be deficient in the catalysis of DHK. As known, there is no DHM accumulated in Arabidopsis due to lacking of flavonoid 3′, 5′-hydroxylase (F3′5′H), whether FhDFR1, FhDFR2, FhDFR3 could accept DHM as substrate should be further validated by the examination of their catalytic properties. Based on the in vitro biochemical assays, we found that all the three FhDFR proteins aforementioned showed a high catalytic efficiency for DHM, whereas only FhDFR2 was proved to convert DHQ to LEUC, which was not consistent to the results of the Arabidopsis tt3-1 mutant complementation mentioned above, this contradiction might be ascribed to the lower catalytic efficiency of FhDFR1 and FhDFR3, which was lost during the preparation and purification of the recombinant proteins. In conclusion, FhDFR1, FhDFR2, and FhDFR3 could utilize both DHQ and DHM as substrate, with a higher activity toward DHM than DHQ.

Based on the metabolites found in the flowers, the anthocyanin biosynthetic pathway in freesia was proposed earlier (Sun et al., 2016). All of the six basic anthocyanin aglycons could be synthesized except pelargonidin derivatives, and the most abundant anthocyanins were delphinidin derivatives. On the contrary, flavonol analysis showed predominant kaempferol glycosides and minor quercetin glycosides, whereas myricetin glycosides were undetectable throughout the flower development (Sun et al., 2016). Therefore, as all the precursors of pelargonidin, cyanidin and delphinidin were present in F. hybrida, the lack of pelargonidin might be ascribed to the substrate specificity of the FhDFR proteins. In addition, it seemed reasonable to deduce that the lack of myricetin in F. hybrida flowers might result from the substrate selectivity of FLS (Figure 8). Thus, it was interesting to conclude that FLS and DFR competed for common substrates in order to direct the biosynthesis of flavonols and anthocyanins, respectively, which was also illustrated by Luo et al. (2015) earlier. Actually, several other plant species, such as Petunia hybrida, Cymbidium hybrida, Angelonia angustifolia, Agapanthus praecox ssp. orientalis (Leighton), have also been found unable to produce pelargonidin derivatives because of the DFR substrate specificities (Mol et al., 1998; Johnson et al., 1999; Gosch et al., 2014; Mori et al., 2014). Therefore, it can be concluded that substrate specificities of FhDFR proteins played crucial role in determination of anthocyanin aglycons in F. hybrdia.

The DFRs are key enzymes in flavonoid biosynthesis. Variable DFR–like gene numbers were therefore found in various genomes, e.g., a single copy DFR is present in Arabidopsis thaliana, in which the anthocyanin and proanthocyanidin components seemed to be simple as well, while multicopy DFR genes exist in M. truncatula, L. japonicus, Populus trichocarpa, and F. hybrida, these metabolites showed more complicated patterns and more diverse physiological functions (Østergaard et al., 2001; Xie et al., 2004; Shimada et al., 2005; Huang et al., 2012). Susumu Ohno hypothesized that gene duplication drives the evolution of novel functions, and deduced three kind fates of the duplicated genes: silence, neofunctionalization and subfunctionalization (Epstein, 1971). Based on the phylogenetic position of DFR, ANR, FNR, and CCR, it can be deduced, that they might have diverged from the same ancestral gene after gene or genome duplication during plant evolution. It could also be expected that some DFR isozymes might be specialized to anthocyanin synthesis, proanthocyanidin or other branch pathways in the bioysnthesis of flavonoids. Huang et al. (2012) found that overexpressing PtrDFR1 in Chinese white poplar (Populus tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation. In addition, the different paralogs might be regulated differentially with spatial and temporal manner under exogenous and endogenous cues. In this study, the expression of both FhDFR1/FhDFR2 and FhDFR3 was testified to be controlled by a common MBW complex, including MYB and bHLH regulators. However, both FhDFR1/FhDFR2 and FhDFR3 could be activated by FhGL3L in combination with the MYB protein AtPAP1, whereas FhTT8L could only regulate FhDFR3 in the presence of AtPAP1. The endogenous FhMYB regulators still need to be further investigated whether the phenomena simply resulting from the heterogenous AtPAP1 or from the evolutionary divergence between FhDFR1/FhDFR2 and FhDFR3.

Previous studies showed that gene duplication acted as a driver for plant morphogenetic evolution, and have possibly allowed the adaptation of the enzymes for specialized functions and contributed to the divergence of plant metabolisms (Rensing, 2014). In this study, we found that the duplicated FhDFR genes from F. hybrida have evolved divergently with different nucleotide sequences and expression patterns. FhDFR1, FhDFR2, and FhDFR3 were involved in the biosynthesis of flavonoinds and determined the components of the anthocyanins in F. hybrida. Comparatively speaking, FhDFR3 might perform more important roles in the biosynthesis of proanthocyanidins. Moreover, they were controlled by a potential MBW complex responsible for anthocyanin biosynthesis. Taken together, the results are not only helpful for future research on DFR evolution and divergence analysis but also useful for manipulating flavonoid biosynthesis in F. hybrida as well as in other monocotyledonous ornamental plants.

YL, XL, XC, XS, and RG performed most of the experiments. SY and TH helped in seedling planting and sample preparation, SW helped analyze the results, XG designed the experiments, wrote and edited the manuscript. LW helped design the experiments and revise the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.