Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes
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- Nakatsuka, T., Abe, Y., Kakizaki, Y. et al. Plant Cell Rep (2007) 26: 1951. doi:10.1007/s00299-007-0401-0
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Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully.
KeywordsFlower colorGenetic engineeringPelargonidinRNAiTobacco
Cauliflower mosaic virus
High performance liquid chromatography
Open reading frame
Flower color is one of the most important characters in floricultural plants. There are various flower colors in the plant kingdom, and most popular floricultural plants have a large number of cultivars with variation in flower coloration through breeders’ efforts. However, some important colors are still unavailable in certain plants. For example, vivid red-colored flowers do not exist in some important floricultural plants, such as petunia, delphinium, Easter lily and gentian. It is difficult to create novel flower colors in such floricultural plants by conventional breeding because suitable genetic resources are absent in related species.
Flower colors are a result of the accumulation of secondary metabolites such as anthocyanins, carotenoids or betalains, and the types of the accumulated pigments largely depend on plant species. Among them, anthocyanins are widely distributed in higher plants and have been well characterized for their structure and biosynthesis (reviewed by Grotewold 2006). They are composed of three basic skeletons, which have a different number of hydroxyl groups at the B-ring, namely pelargonidin, cyanidin and delphinidin. Pelargonidin derivatives generally have the lower absorption spectra and can contribute to vivid red-flower color. The biosynthesis of anthocyanidins (aglycons) is regulated by two processes, namely the hydroxylation of the B-ring, and the substrate specificity of dihydroflavonol 4-reductase (DFR) (Forkmann and Ruhnau 1987). With regard to the former, dihydrokaempferol (DHK) is converted to dihydroquercetin (DHQ) or dihydromyricetin (DHM) by flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′,5′H), respectively. In the latter case, it is considered that DFR in some species, such as petunia (Forkmann and Ruhnau 1987; Johnson et al. 2001), cymbidium (Johnson et al. 1999) and gentian (Tanaka et al. 1996), cannot reduce DHK efficiently as a substrate. Thus, such plant species cannot produce pelargonidin-based orange to red colors in their flowers even if F3′H and F3′,5′H activities are absent (Forkmann and Ruhnau 1987; Brugliera et al. 1999). In such cases, molecular engineering such as genetic transformation technology might create solutions for the production of new flower colors (Tanaka et al. 1998, 2005; Mol et al. 1999; Forkmann and Martens 2001).
Meyer et al. (1987) first demonstrated the successful engineering of pelargonidin synthesis by the expression of the maize DFR (A1) gene in petunia to produce brick-red flowers. A DFR gene from gerbera and rose also could enhance the formation of pelargonidin pigments in transgenic petunia plants (Helariutta et al. 1993; Tanaka et al. 1995). These studies have used as a transformed host the DHK-accumulated petunia triple mutant, which is deficient in F3′H (ht1) and F3′,5′H (hf1 and hf2) enzymatic activities. However, such a triple-mutant line is unavailable in many ornamental flowers. Even if the individual mutant lines exist, it is difficult to accumulate several mutations in one genotype in many ornamental plants, because they are highly heterologous and in some cases clonal varieties are bred. To overcome these problems, a genetic transformation technique is the most convenient to regulate multiple flavonoid biosynthetic genes without the use of flavonoid biosynthetic-deficient mutants.
During the past few decades, many researchers have reported some useful molecular techniques for down-regulating gene expression, including antisense, co-suppression and RNA interference (RNAi) technology (van der Krol et al. 1988; Napoli et al. 1990; Waterhouse et al. 1998). RNAi could induce more efficient and stronger gene silencing than the other two technologies (Waterhouse et al. 1998; Chuang and Meyerowitz 2000). RNAi-mediated gene silencing is receiving widespread use for down-regulation of endogenous gene expression in many eukaryotic organisms, including the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and a variety of mammals and plants (reviewed by Hannon 2003; Abbott 2006; Couzin 2006). RNAi has been applied to suppress some structural genes in anthocyanin biosynthesis, causing the inhibition of anthocyanin accumulation and a change in flower color in transgenic plants (Fukusaki et al. 2004; Tsuda et al. 2004; Nishihara et al. 2005; Nakamura et al. 2006). Recently, Miki et al. (2005) have developed novel RNAi constructs, which could induce simultaneous silencing of up to four genes, using chimeric triggers of OsRac genes. We adopted this silencing method for flower color modification in this study.
Here we report the successful modification of flower color by the combination of suppression of two endogenous genes and overexpression of a foreign gene in wild-type tobacco plants. This system provides a useful and powerful tool for metabolic engineering of not only flower color, but also a diversity of other complex biosynthetic pathways.
Materials and methods
Vector construction and tobacco transformation
An open reading frame (ORF) of the DFR gene from Gerbera hybrida (Helariutta et al. 1993; accession number Z17221) under the control of the CaMV35S promoter was inserted into a binary vector harboring the hygromycin-resistant gene (hpt) to produce the plasmid pEBisHR-35SgerberaDFR (Fig. 1b). Both the 35Spro-intNtFLS:NtF3′Hir cassette and the 35Spro-gerbera DFR cassette were inserted into a binary vector pEBisBR harboring the bar gene (pEBisBR-35SgerberaDFR-35SintFLS:F3′Hir, Fig. 1c). Each binary vector was transformed into Agrobacterium tumefaciens EHA101 or EHA105 and used for tobacco transformation. Tobacco plants that were aseptically grown from seeds for about one month were transformed via an A. tumefaciens-mediated leaf disc procedure (Horsch et al. 1985), and selected using a medium containing either 5 mg/l of bialaphos or 30 mg/l of hygromycin depending on the binary vector. After rooting and acclimatization, the regenerated plants were grown in a greenhouse to set seeds by self-pollination. A T1 transformation line no. 9 (pEBis35SintNtF3′Hir-bar) was reinoculated with pEbisHR-35SgerberaDFR and selected using 30 mg/l of hygromycin. The seeds of transgenic T1 plant lines were sown on a medium containing 5 mg/l of bialaphos and/or 30 mg/l of hygromycin. Resistant plants were transferred to a closed greenhouse to produce flowers and for use in further analyses.
Northern blot analysis
Total RNA was isolated from wild-type and transgenic tobacco at flower developmental stage 3, as defined by Nishihara et al. (2005), using the FastRNA pro GREEN Kit (Q-gene, CA, USA). Five micrograms of total RNAs were subjected to northern blot analysis. Probes for NtF3′H, NtFLS and gerbera DFR genes, were prepared with the PCR-DIG Probe Synthesis Kit (Roche Diagnostics, Germany). Hybridization and detection were performed as described previously (Nakatsuka et al. 2005).
Flavonoid compound analyses
To measure anthocyanidin and flavonol compositions in transgenic plants, anthocyanin and flavonol pigments were extracted from a single petal of each transformant with 1 ml methanol containing 1% HCl. To each extract 4.5 ml 3N HCl was added and the extracts were boiled for 90 min for hydrolysis. For anthocyanidin samples, the hydrolyzed solutions were extracted twice with 1 ml isoamylalcohol. The isoamylalcohol fractions were evaporated and redissolved in 500 μl methanol containing 0.1% HCl. For flavonol samples, the hydrolyzed solutions were extracted twice with 5 ml ethyl acetate. The alcohol fractions were evaporated and redissolved in 500-μl methanol. High performance liquid chromatography (HPLC) analyses were performed as described by Nakatsuka et al. (2006).
Vector construct and tobacco transformation
Transgenic tobacco plants were regenerated on the selective media by inoculation of leaf sections with A. tumefaciens harboring each binary vector as illustrated in Fig. 1. For all transformation experiments, more than 10 independent transgenic tobacco lines were produced. After self-pollination, T1 transgenic plants that were considered to have a single copy of the T-DNA insertion by segregation analyses were selected and subjected to further analysis.
Phenotype of transgenic tobacco plants
Although F3′Hir transgenic tobacco flowers showed decreased anthocyanin accumulation, no effect on flower hue was observed. We hypothesized that the endogenous tobacco DFR could not reduce DHK as a precursor of pelargonidin. Because clone no. 9 displayed the strongest suppression phenotype among F3′Hir-transgenic lines (Fig. 3b), we chose this clone as a host for inoculation with pEBisHR-35SgerberaDFR (Fig. 1b), harboring the expression cassette of a DFR ORF from G. hybrida under the control of the CaMV35S promoter. It is well known that gerbera DFR (GhDFR) efficiently converts DHK to leucopelargonidin (Helariutta et al. 1993). Of 10 transformants, termed F3′Hir+GhDFRox, 3 transformants (clone nos. 2, 3 and 10) were estimated to have a single copy of the gerbera DFR gene in their genomes by segregation analysis (data not shown). The three F3′Hir+GhDFRox transformant lines exhibited a slight enhancement of flower color intensity and change in hue (Fig. 3c) compared with the original F3′Hir transformants (Fig. 3b).
Although the flowers of F3′Hir+GhDFRox transgenic tobacco plants had modified color hues, the visual effect was not so intense as to generate a red color. This is because DHK was metabolized predominantly by the endogenous tobacco FLS rather than by the foreign gerbera DFR. Therefore, the suppression of the FLS gene also seemed to enhance flower pigmentation in addition to suppression of the F3′H gene. We employed the technology that can suppress the expression of multiple genes simultaneously by a single construct (Miki et al. 2005). This construct harbored two cassettes expressing an inverted repeat of tandem orientations of NtFLS and NtF3′H partial fragments and overexpressing a gerbera DFR gene, termed FLS:F3′Hir,GhDFRox (Fig. 1c).
Of 16 FLS:F3′Hir,GhDFRox transformants, 6 transgenic lines were estimated to have a single copy of the T-DNA insertion in their genomes. Of the six transformant clones, three clones, nos. 4, 15 and 16, clearly showed red coloration and an increase in color intensity in their flowers (Fig. 3d-f, respectively). In addition, ectopic pigmentations were observed in the corolla tubes and filaments of the transgenic lines (Fig. 3g, i). These three lines were subjected to further analyses.
Other than the modification of flower color, no other visible change was observed in all transgenic tobacco plants produced in this study.
Expression analysis of NtF3′H, NtFLS and GhDFR in transgenic tobacco plants
Anthocyanidin and flavonol analyses by HPLC
Tobacco flowers abundantly accumulated flavonol derivatives as colorless flavonoids together with colored anthocyanins. Flavonol composition of the petals in transgenic plants was also measured by HPLC analysis (Fig. 5b). In wild-type flowers, mainly quercetin and kaempferol derivatives accumulated, and the ratio of kaempferol derivatives to quercetin derivatives was two-thirds. The profiles of flavonol composition in F3′Hir and F3′Hir+GhDFRox transgenic plants were inversely related to those of wild type. Namely, three F3′Hir line nos. 2, 3 and 10 showed markedly increased kaempferol levels and decreased quercetin levels. Three FLS:F3′Hir,GhDFRox transformants line nos. 15 and 16 showed a modest decrease in levels of quercetin derivatives and a slight increase in kaempferol derivatives. In contrast, quercetin and kaempferol levels were reduced notably in clone no. 4, probably resulting from the strong reduction of NtFLS activity.
Tobacco plants predominantly accumulate cyanidin derivatives in their petals as anthocyanins (cyanidin 3-rutinoside; Aharoni et al. 2001). On its own, the overexpression of gerbera DFR, which can utilize DHK as a substrate, did not cause a change in flower color in tobacco (data not shown). This suggested that it is necessary to modify the flavonoid biosynthetic pathway for the production of pelargonidin pigments in tobacco. NtF3′H-downregulated (F3′Hir) transgenic tobacco exhibited reduced cyanidin accumulation in their petals, but never enhanced levels of pelargonidin pigments (Figs. 3b, 5a). In petunia, torenia and Osteospermum hybrida, suppression of the F3′H or F3′,5′H genes did not cause significant accumulation of pelargonidin derivatives (Suzuki et al. 2000; Tsuda et al. 2004; Seitz et al. 2007). In addition, since the overexpression of F3′H from gentian in tobacco led to enhanced cyanidin accumulation (Nakatsuka et al. 2006), these results revealed that F3′H is critical enzyme for controlling the amounts of cyanidin derivatives in tobacco flowers.
We combined downregulation of the NtF3′H gene with overexpression of the gerbera DFR gene, and this caused transgenic tobacco F3′Hir+GhDFRox lines to produce additional pelargonidin pigments (Figs. 3d–f, 5a). In petunia, suppression of the F3′H gene by antisense or RNAi combined with over expression of rose DFR caused accumulation of pelargonidin derivatives as the major flower pigments (Tsuda et al. 2004). Our results revealed that tobacco DFR could not catalyze efficiently the reduction of DHK as a substrate, and that the tobacco flower could produce small amounts of pelargonidin pigments after introduction of a foreign DFR gene. However, in F3′Hir+GhDFRox transgenic tobacco lines, the level of additional pelargonidin accumulation in the transgenic flowers was insufficient to change the flower color dramatically (Fig. 2c). DHK is a substrate of both F3′H and DFR enzymes, but is also a substrate of FLS, which converts DHK to the flavonol kaempferol. Therefore the excessive DHK is considered to be utilized in flavonol biosynthesis in the flowers of F3′Hir+GhDFRox transgenic tobacco plants (Figs. 2, 5b). Although F3′Hir+GhDFRox transgenic tobacco flowers showed an increase in the level of kaempferol derivatives and a decrease in quercetin derivatives, the level of cyanidin derivatives was not enhanced. In previous studies that successfully used petunia triple mutants, the deficiency in FLS activity was not necessary for production of pelargonidin pigments (Tsuda et al. 2004). It is likely that tobacco FLS has more efficient catalytic activity for DHK as a substrate than gerbera DFR and petunia FLS. Therefore, the suppression of FLS activity was considered to be necessary to produce pelargonidin pigments abundantly in tobacco flowers. When the expression of F3′H and FLS was downregulated by chimeric RNAi, and gerbera DFR was over expressed by a single construct (FLS:F3′Hir,GhDFRox), red-flowered phenotypes could be produced successfully in transgenic tobacco flowers (Fig. 3d–f). Anthocyanins composed of pelargonidin and cyanidin, and the accumulation of cyanidin derivatives was greater than pelargonidin derivatives in the flowers of FLS:F3′Hir,GhDFRox transgenic tobacco despite the reduced production of F3′H transcripts. In petunia and lisianthus, the suppression of FLS transcripts could lead to enhance accumulation of anthocyanin (Nielsen et al. 2002; Davies et al. 2003). Since the flowers of F3′Hir transgenic tobacco plants also exhibited pale pink colors, it was thought that the DHK pool, expanded by the suppression of FLS activity, was converted efficiently by the remaining F3′H activity. By the enhancement of anthocyanin accumulation in FLS:F3′Hir,GhDFRox transgenic tobacco, the corolla tubes and filaments became pigmented, whereas few accumulating pigments were found in these organs in wild-type tobacco plants (Fig. 3h–i).
The substrate specificity of DFR enzymes is one of the most important factors in the formation of pelargonidin pigments. Previous studies have isolated the genes encoding DFR, which can catalyze the reduction of DHK, from several cultivated plants including maize (Schwarz-Sommer et al. 1987), gerbera (Helariutta et al. 1993) and rose (Tanaka et al. 1995), and resulted in brick-red flowers when introduced into petunia mutants. Petunia DFR cannot accept DHK as a substrate to produce pelargonidin and contains an aspartic acid (Asp) residue at the 134th position, whereas DFR that can reduce DHK in several other plants contains an asparagine (Asn) residue (Beld et al. 1989; Johnson et al. 2001). We isolated two tobacco DFR cDNA expressed in flowers, and the deduced amino acid sequence of the DFR also contained an Asp residue at this position (Supplemental Fig. S2). This study used a DFR gene from G. hybrida for pelargonidin formation in tobacco, because the gerbera DFR gene showed stronger and more stable expression than the maize A1 gene in transgenic petunia (Elomaa et al. 1995). The choice of the gene source is an important factor to be considered in molecular engineering even if the gene encodes the same enzyme activity (Tanaka et al. 1998). This also suggested that it is difficult for the enzyme-encoding transgene to compete with endogenous enzymes and intracellular conditions. It might be possible to induce tobacco endogenous DFR suppression to prevent cyanidin accumulation in future studies. In addition, production of transgenic tobacco plants that have other gene combinations such as NtFLSir+GhDFRox and NtFLSir alone will also give helpful information to elucidate the metabolic flux of flavonols and anthocyanins in tobacco flowers.
We used RNAi technology for gene silencing in tobacco plants. RNAi techniques have been shown to induce efficient suppression of the targeted gene in plants and several eukaryotic organisms, in comparison with antisense and co-suppression technologies (Waterhouse et al. 1998; Chuang and Meyerowitz 2000). Of structural genes in the flavonoid biosynthetic pathway, the CHS and CHI genes were strongly suppressed by RNAi in transgenic torenia and tobacco plants, respectively, and displayed a decrease in flower color intensity (Fukusaki et al. 2004; Nishihara et al. 2005). In this study, we employed the RNAi technique using chimeric triggers, because the available promoter might be limited in many floricultural plants. Usually the silencing of two genes was necessary to introduce the two inverted repeat structures under the control of independent promoters. Miki et al. (2005) have reported that inverted repeats fusing the multiple triggers could down-regulate the expression of multiple genes of the OsRac family. Thus, we attempted to suppress the expression of both F3′H and FLS in transgenic tobacco. FLS:F3′Hir,GhDFRox transgenic tobacco plants showed that fused triggers could suppress the expression of both genes in their flowers (Fig. 4). However, the suppressive ability of the FLS:F3′Hir construct was slightly reduced in comparison with that of a single trigger (F3′Hir) judging by the amounts of cyanidin derivatives. In rice, the suppression efficiency was also reduced when the number of fused triggers increased (Miki et al. 2005). It might be a result of the instability of the long hairpin RNA structure (>1 kb). Moreover, northern blot analysis highlighted the difference in degradation of NtF3′H transcripts observed between F3′Hir- and FLS:F3′Hir-transformants (Fig. 4). Preliminary experiments showed that F3′H siRNA was detected in four F3′Hir-transformants but not in all FLS:F3′Hir,GhDFRox transformants (data not shown). Therefore, the chimeric structure of triggers might affect the efficiency of gene silencing and the RNAi mechanism even if the trigger of NtF3′H has the same sequence and length. A detailed analysis is necessary to optimize the sequence length and position for efficient suppression of multiple genes.
In conclusion, we demonstrated a simple and efficient method to produce red-flowered plants by molecular engineering using tobacco as a model plant. In the case of other floricultural plants, some modifications might be necessary, such as changing the endogenous genes targeted and the promoters to accumulate DHK pools. Currently, we are attempting to apply this technology to some other ornamental plants.
We thank A. Kubota, R, Horikiri, R. Takahashi and A. Oikawa, Iwate Biotechnology Research Center (IBRC), for technical assistance.