Molecular characterization of mutations in white-flowered torenia plants
© Nishihara et al.; licensee BioMed Central Ltd. 2014
Received: 27 January 2014
Accepted: 20 March 2014
Published: 2 April 2014
Torenia (Torenia fournieri Lind.) is a model plant increasingly exploited in studies in various disciplines, including plant engineering, biochemistry, physiology, and ecology. Additionally, cultivars with different flower colors have been bred and made commercially available. Flower color in torenia is mainly attributed to the accumulation of anthocyanins, but the molecular mechanisms inducing flower color mutations in torenia have not been well elucidated. In this study, we therefore attempted to identify the cause of white coloration in torenia by comparing the white-flowered cultivar Crown White (CrW) with Crown Violet (CrV), a violet-flowered variety.
In an expression analysis, no flavanone 3-hydroxylase (TfF3H) transcript accumulation was detected in CrW petals. Sequence analyses revealed that a novel long terminal repeat (LTR)-type retrotransposable element, designated as TORE1 (Torenia retrotransposon 1), is inserted into the 5′-upstream region of the TfF3H gene in CrW. A transient expression assay using torenia F3H promoters with or without TORE1 insertion showed that the TORE1 insertion substantially suppressed F3H promoter activity, suggesting that this insertion is responsible for the absence of F3H transcripts in white petals. Furthermore, a transformation experiment demonstrated that the introduction of a foreign gentian F3H cDNA, GtF3H, into CrW was able to recover pink-flower pigmentation, indicating that F3H deficiency is indeed the cause of the colorless flower phenotype in CrW. Detailed sequence analysis also identified deletion mutations in flavonoid 3′-hydroxylase (TfF3′H) and flavonoid 3′,5′- hydroxylase (TfF3′5′H) genes, but these were not directly responsible for white coloration in this cultivar.
Taken together, a novel retrotransposable element, TORE1, inserted into the F3H 5′-upstream region is the cause of deficient F3H transcripts in white-flowered torenia, thereby leading to reduced petal anthocyanin levels. This is the first report of a retrotransposable element involved in flower color mutation in the genus Torenia.
KeywordsTorenia fournieri F3H Muation LTR-type retrotransposon White flower
Natural or spontaneous mutations, which accelerate evolution in living organisms, have a number of causes. In flowering plants, flower color mutations occur widely in nature, and floricultural plants in a variety of flower colors have been produced through artificial selection of this natural variability. Flavonoids, such as flavones, aurones, flavonols, and anthocyanins, are the most important plant pigments associated with flower coloration. The flavonoid biosynthetic pathway is one of the most extensively studied pathways in plant specialized metabolism . The biosynthetic genes necessary for biosynthesis of colored anthocyanins have been especially well studied in many plant species, including, Arabidopsis, snapdragon, petunia, grape, and maize, and have become targets for molecular breeding [1–4]. In addition to flower coloration, studies of anthocyanin-based coloration and associated mutations in fruits, seeds, and other organs have provided information helpful for elucidation of flavonoid biosynthesis-related genes. In particular, recent molecular biological investigations have revealed the basic mechanisms generating these mutations and their effects on pigmentation at the molecular level in different plant species.
Torenia (Torenia fournieri Lind., also known as wishbone flower) is a perennial plant widely used as a bedding flower from early spring through summer. Cultivars in different flower colors, such as white, blue, and pink, have been generated by conventional breeding and are now commercially available. Because of its various merits in regard to molecular analyses (reviewed in [5, 6]), the species is proposed as a potential new model flower to study a wide range of floral traits. Torenia fournieri and T. hybrida (T. fournieri × T. concolor) are frequently used in transgenic studies of various subjects such as flower color [7–9], anthocyanin synthesis , chlorophyll degradation , flower shape [11–14], and fertilization . Among studied characteristics, flower color is one of the most targeted traits in molecular investigations, as flower color modification is of strong interest from both basic and applied research perspectives. Torenia flower pigments are composed of flavonoid anthocyanins. Chemical analysis has revealed the anthocyanin components of T. fournieri ‘Crown Violet’  and T. hybrida ‘Summerwave Blue’ . Using these cultivars, flower colors ranging from the original violet to white, pink, and yellow have been successfully produced by genetic engineering of flavonoid biosynthetic genes [7–9, 16–19].
As mentioned above, variously flower-colored torenia cultivars and lines have been produced by both conventional and molecular methods, but the origins of color mutations in the breeding materials are largely unknown. Because they are easily recognizable by eye and hence good plant research materials, flower color mutations have been studied extensively in species such as petunia [20, 21], snapdragon [22, 23], and morning glory [24, 25]. For example, the variegated-flower morning glory mutation is caused by the insertion of the Tpn1 transposable element into the dihydroflavonol 4-reductase (DFR) gene , and white-flowered morning glory is derived from insertions of Tpn1-related DNA transposable elements into an intron of the chalcone synthase (CHS) gene . Red-flowered phenotypes in commercial petunias have been attributed to transposon insertion mutations of the F3′5′H gene . Similarly, various mutations related to flower pigmentation have been studied in carnation [29–31], with class I, class II, and other transposable elements implicated in the color changes. We have also identified several DNA- and RNA-type transposable elements in white- or pink-flowered mutants in Japanese gentian flowers [32–34]. With respect to torenia, little is known regarding the source of flower color mutations, with one exception: Nishijima et al.  recently reported that a ‘flecked’ mutant, bearing variegated flowers, originated through the insertion of an Enhancer/Suppressor-Mutator (En/Spm)-like transposon (Ttf1) into the intron of the TfMYB1 transcription factor gene. The causal agents of flower color mutations in other torenia cultivars have not yet been identified.
In the present study, we uncovered and analyzed mutations in a white-flowered torenia through comparison with a violet-flowered cultivar. We first identified a novel retrotransposable element, designated TORE1, in torenia based on a molecular biological approach. Using both transient and stable transformation experiments, we then confirmed that the insertion of TORE1 into the flavanone 3-hydroxylase (F3H) promoter region contributes to the white-flowered phenotype. We found two additional mutations in this cultivar and considered their association with white coloration. This is the first known identification of a retrotransposable element in the genus Torenia.
Analysis of flavonoid components in torenia flowers
Expression analysis of flavonoid biosynthetic genes in torenia flowers
Identification of genomic and cDNA sequences of three flavonoid biosynthetic genes in torenia plants
Based on the results of the pigment and expression analyses, we speculated that three genes, F3H, F3′H, and F3′5′H, were causally related to the CrW flower color mutation. We therefore compared genomic sequences of these three genes between the two cultivars. We generated DNA fragments that spanned start and stop codons using genomic PCR, and analyzed the sequences. F3H was identical between CrV and CrW, whereas deletion mutations were present in F3′H and F3′5′H genomic sequences in both CrV and CrW (Additional file 2: Figure S2). In particular, a 12-bp deletion was observed in exon 3 of the CrW F3′H gene, presumably lacking four amino acid residues (Additional file 2: Figure S2A). The F3′5′H gene from CrW also had a single-base (cytosine) deletion in exon 1, resulting in a frame shift (Additional file 2: Figure S2B). These mutated F3′H and F3′5′H sequences were also present in CrV, indicating that CrV is heterozygous for both gene mutations.
Isolation of the 5′-upstream region of the F3Hgene from torenia plants
Southern blot analysis of three flavonoid biosynthetic genes and TORE1in torenia
Transient expression analysis for F3Hpromoter activity
Flower color complementation in F3H-overexpressed transgenic torenia plants
In this study, expression analysis of flavonoid biosynthetic genes revealed that only the expression of F3H was completely reduced in CrW. F3H is a 2-oxoglutarate-dependent dioxygenase that acts in the flavonoid biosynthetic pathway to hydroxylate flavanones to dihydroflavonols, which are common precursors for major classes of flavonoids, flavonols, catechins, anthocyanins, and proanthocyanidin in plants [1, 39]. The gene encoding F3H cDNA was first isolated from petunia , with homologous genes subsequently cloned in many plant species. F3H gene mutations affecting color pigmentation have been reported in various plant species, including petunia , soybean , Japanese morning glory , and carnation . Because anthocyanins are synthesized from dihydroflavonols, reduced F3H expression is the likely cause of the torenia CrW colorless flower phenotype.
Analysis of genomic structures did not uncover any mutations in either coding or intron regions of torenia F3H, but a long insertion (TORE1) was found in the 5′-upstream region of F3H in CrW. This insertion was identified as a retrotransposable element of the LTR subtype [44, 45]. TORE1 has typical characteristics of LTR-type retrotransposons, namely 560-bp LTRs with 5 bps of target site duplication (TSD), a PBS, and a PPT, and encodes a partial gag-pol protein (Figure 3; Additional file 2: Figure S3). TORE1 may thus be a nonautonomous element derived from an originally autonomous one. Southern blot analysis also indicated the presence of TORE1-like elements in the torenia genome.
Transient expression analysis using various constructs of the F3H promoter revealed that the TORE1 insertion is indeed involved in reduced promoter activity (Figure 5). For this analysis, we used the heterologous transcription factor genes GtMYB3 and GtbHLH1, which regulate flavonoid biosynthesis in gentian flowers. These two genes were chosen because they are well-characterized and constitute a reliable transient assay system established in our previous studies [33, 38]. Although torenia endogenous TfMYB1 and TfbHLH1 genes that are probably responsible for regulation of anthocyanin biosynthesis have been recently isolated, the evidence for TfMYB1-TfbHLH1 interaction leading to activation of flavonoid biosynthetic genes remains inconclusive. Further analysis is therefore required to clarify the effects of TfMYB1 and TfbHLH1 transcription factor genes on activation of the torenia F3H promoter.
Many examples of transposable elements affecting gene expression have been noted in various organisms, with different classes of active transposons observed in genomes of eukaryotes including plants [46, 47]. Transposable elements have been confirmed to contribute to plant evolution and diversity . Retrotransposon or retrotransposon-like sequences, in particular, are ubiquitous components of plant genomes, and their impacts on plant genome structure and function have also been revealed (reviewed in ). Transposon insertions have been found in all types of genomic sequences, including coding (exon) and noncoding (intron, UTR, upstream, and downstream) regions. In a previous study, an En/Spm-like transposon (Ttf1) insertion in the TfMYB1 second intron was found to cause a mutant torenia phenotype by reducing expressions of flavonoid biosynthetic genes, including CHS, F3H, DFR, ANS, and UDP-glucose: flavonoid glucosyltransferase (UFGT) . In our case, the insertion of TORE1 completely suppressed F3H expression, as shown by RT-PCR and northern blot analysis. Transposable element insertions often induce epigenetic changes, such as DNA methylation, that cause strong gene silencing [47, 50]; this fact suggests that TORE1 may reduce F3H expression through a similar silencing mechanism. In fact, Southern blot analysis of CrW genomic DNA digested with Hind III revealed some larger bands when probed with F3H. Hind III is sensitive to cytosine methylation within the recognition site, and slowly cleaves hemimethylated AAGm5CTT . Partial digestion with Hind III due to the effect of de novo methylation has also been reported in transgenic pea plants showing inducible co-suppression of the transgene after virus infection . Such an epigenetic change probably contributes, along with disruption of the F3H promoter, to the reduction in F3H promoter activity. Further studies using methylation-sensitive restriction enzymes or bisulfite sequencing would confirm this hypothesis.
Detailed examination of the 5′-upstream region of the F3H promoter in CrW genomic DNA revealed the presence of a solo-LTR insertion in addition to the full-length TORE1 insertion (Figure 3; Additional file 2: Figure S4). Southern blotting generated a banding pattern inconsistent with the presence of the solo LTR in the F3H promoter, indicating that the TORE1-inserted F3H and solo LTR-inserted F3H are not allelic. The solo LTR is probably derived from a recombination between LTRs within TORE1 in certain cells. As an example, the formation of solo LTRs through unequal homologous recombination of two LTRs has been previously reported in rice . Based on the presence of solo LTRs and variously truncated fragments in plant genomes, unequal homologous recombination and illegitimate recombination have been inferred to drive genome size decreases in Arabidopsis  and rice . CrW sometimes shows faint pink-recovered sections in flower petals (Additional file 2: Figure S8). A TORE1 excision event is probably not involved in this recovery, however, as class I transposons move by a "copy and paste" process. In the transient expression assay, the solo-LTR insertion was less effective in reducing promoter activity than was the full TORE1 insertion (Figure 5). This result suggests that the advent of the solo LTR is responsible for the partially recovered phenotype. Further studies are needed to determine the mechanism and developmental timing of this TORE1 recombination event in the F3H promoter of CrW torenia plants.
The white coloration of CrW can clearly be attributed to the F3H mutation, a conclusion confirmed by the complementation study (Figure 6). This finding is further supported by the fact that knockdown of torenia F3H by RNAi produces white-flowered torenia plants in the blue-flowered cultivar Summerwave Blue . Nevertheless, mutations were also found in F3′H and F3′5′H gene sequences in CrW (Additional file 2: Figure S2). Deficiency of flavonoid B-ring hydroxylation activities is suggested by two observations: 1) apigenin was the only flavone accumulating in CrW petals (Figure 1) and 2) F3H-overexpressing transgenic CrW lines accumulated pelargonidin derivatives, not delphinidin derivatives (Additional file 2: Figure S7). F3′H and F3′5′H genes in CrW have deletion mutations that should affect enzyme activities. In particular, the single-base deletion in exon 1 of F3′5′H results in a frameshift and subsequent premature termination of the enzyme (Additional file 2: Figure S2). Such mutations are frequently observed in other plants. In soybean, for example, single-base deletions in F3′H and flavonol synthase (FLS) are associated respectively with gray pubescence color  and magenta flower color . A 4-bp insertion mutation in UDP-glucose:anthocyanidin 3–O-glucoside-2''–O-glucosyltransferase (3GGT) induces a dusky flower color in morning glory . In three morning glory species, a single C to T base transition, a single T insertion, and insertion of the transposable element Tip201 in F3′H are reportedly involved in reddish flowers . In the case of CrW, the 12-bp deletion in exon 3 of F3′H did not cause a frameshift, but apparently resulted in the absence of four amino acid residues (Additional file 2: Figure S2). This deleted amino acid sequence partially overlapped with a threonine-containing binding pocket (A/G-G-X-D/E-T-T/S) regarded as an oxygen-binding motif of cytochrome P450 monooxygenase, required in catalysis . Although in vitro enzyme activity was not examined, the lack of this essential motif is very likely responsible for diminished F3′H enzymatic activity. In addition, CrW exhibits a shorter UV maximum absorption peak—333 nm compared with 346 nm in CrV—and recovered anthocyanin pigmentation in F3H-overexpressing CrW petals are pink, a color arising from pelargonidin derivatives that lack B-ring hydroxylation (Additional file 2: Figure S7). These results also indicate the absence of F3′H and F3′5′H enzyme activities. It is unlikely, however, that these F3′H and F3′5′H mutations are responsible for white coloration in CrW. For example, deficiency of hydroxylation activity in the anthocyanidin B-ring caused a color shift, but not to white as reported previously [56, 59]. How CrW came to possess F3′H and F3′5′H mutations is clearly unknown, but they probably arose in this cultivar during breeding selection for flower colors such as pink, lavender, and blue. Another possibility is that the breeding parents of CrW contained the latent mutations of these two genes. Torenia plants of the Crown series have various flower colors, and this variation may depend on genotype. In fact, CrV is heterozygous with respect to these two genes. It is also likely that the other flavonoid biosynthetic genes, besides F3′H and F3′5′H, have heterozygous mutations, because torenia is primarily an outcrossing species. Further analysis of other cultivars of the Crown series should also provide clues useful for answering this question.
In summary, we investigated the causal factor(s) of white flower color in Crown White, a torenia cultivar. Using transient and transgenic approaches, we demonstrated that a novel retrotransposable element (TORE1) inserted into the 5′-upstream region of the F3H gene is directly responsible for the colorless flower phenotype. Further investigations will be required to understand the dynamics of TORE1 in the torenia genome. As torenia cultivars have been produced in various flower colors, we expect that additional mutations will soon be uncovered using molecular approaches. We believe that the Crown White cultivar will be useful for genetic engineering of novel flower colors using various plant pigments.
Torenia fournieri plants were used in this study. White-flowered Crown White (CrW) and violet-flowered Crown Violet (CrV) cultivars selected from ‘Crown Mix’ (PanAmerican Seed Company, West Chicago, IL, USA) were kindly provided by Dr. Ryutaro Aida (National Institute of Floricultural Science, Japan). The plants were cultivated in a greenhouse under natural conditions from spring to summer. For transformation experiments, in vitro cultured plants were used as described previously .
Analysis of pigment components in torenia flower petals
Fully opened flower petals of CrV and CrW were used. Flower petals were extracted in 0.1% HCl-methanol with gentle shaking overnight at 4°C. Spectrophotometric analysis was performed using a SpectroMax 190 absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA). Flavonoid aglycones of anthocyanins and flavones in flower petals were analyzed by high-performance liquid chromatography (HPLC) as described previously .
Expression analyses of flavonoid biosynthetic genes
Total RNA was isolated from opening flowers (stage 4 as defined by Ueyama et al. ) using a FastRNA pro GREEN kit (Qbiogene, Irvine, CA, USA). cDNAs were synthesized from total RNA after removal of genomic DNA using a PrimerScript RT reagent kit with gDNA Eraser (Takara Bio, Shiga, Japan). Semi-quantitative RT-PCR analysis was performed using primer sets listed in Additional file 1: Table S1 for nine flavonoid biosynthetic genes from T. hybrida: chalcone synthase (CHS, accession no. AB012923), chalcone isomerase (CHI, AB548584), flavanone 3-hydroxylase (F3H, AB211958), dihydroflavonol 4-reductase (DFR, AB012924), anthocyanidin synthase (ANS, AB044091), flavonoid 3′-hydroxylase (F3′H, AB057672), flavonoid 3′,5′-hydroxylase (F3′,5′H, AB012925), anthocyanin 5-O-glucosyltransferase (5GT, AB076698), and flavone synthase II (FNSII, AB028152). An actin gene (ACT3, AB330989) was used as an endogenous control. Each 50-μL reaction mixture contained 1× Ex Taq buffer, 200 μM dNTPs, 0.5 μM of each primer, 5 units Ex Taq polymerase (Takara Bio), and 1 μL cDNA template. PCR conditions were as follows: 1 min 30 s at 94°C, followed by 26 to 34 cycles of 20 s at 94°C, 40 s at 55°C, and 2 min at 72°C, and a final extension of 10 min at 72°C. The PCR products were separated by electrophoresis on a 1.0% agarose gel in TAE buffer and stained with ethidium bromide.
Total RNAs (5 μg) were subjected to northern blot analysis. Probes for torenia F3H, F3′H, and F3′5′H were prepared using a PCR-DIG Probe synthesis kit (Roche Diagnostics, Basel, Switzerland) using primers pairs listed in Additional file 1: Table S1. Hybridization and detection were performed using a DIG Nucleic Acid Detection kit (Roche Diagnostics).
Determination of torenia F3H, F3′H, and F3′5′Hgene sequences
Genomic DNAs were isolated from leaves of each cultivar using a Nucleon PhytoPure kit (GE Healthcare, Little Chalfont, UK). PCR reactions were performed as described above, except that genomic DNAs were substituted for the cDNA templates. The primer pairs used are listed in Additional file 1: Table S1. The amplified fragments were subcloned into a pCR4TOPO TA cloning vector (Life Technologies, Carlsbad, CA, USA) and sequenced using a BigDye Terminator version 1.1 cycle sequencing kit on an ABI 3130 genetic analyzer (Life Technologies).
Isolation of 5′-upstream regions of the torenia F3Hgene
The 5′-upstream region of the torenia F3H gene was identified using inverse PCR. One microgram of genomic DNA of CrV was digested with the restriction enzyme Hind III and self-ligated using a Takara ligation kit version 3.0 (Takara Bio). Inverse PCR was performed in 25-μl reaction mixtures containing 100 ng ligated genomic DNA, 1× LA buffer, 2.5 mM MgCl2, 400 μM dNTPs, 0.2 μM of each primer, and 1.25 units of LA Taq polymerase (Takara Bio). The primer sets used are described in Additional file 1: Table S1. Reaction conditions consisted of pre-heating at 94°C for 90 s, 35 cycles at 95°C for 20 s, 60°C for 40 s, and 72°C for 3 min, and an extension at 72°C for 10 min. Amplified fragments of about 5 kb for F3H were subcloned and sequenced as described above. The putative transcriptional initiation site was determined by 5′-RACE technology using a GeneRacer kit (Life Technologies). Several primers designed from the sequence of the 5′-upstream region of CrV were used for PCR amplification of CrW. The PCR products were also subcloned and sequenced as described above.
Southern blot analysis of torenia F3H, F3′H, and F3′5′H genes and TORE1
Genomic DNAs (10 μg) were digested with Hind III, Eco RI, or Xba I, followed by separation on 1% agarose gels and transfer to nylon membranes. Coding sequences of F3H, F3′H, and F3′5′H were used as probes. LTR and gag-pol protein sequences were also used. Hybridization and detection was performed as described previously .
Transient expression assays of F3Hpromoter activities using Arabidopsis suspension cells
Arabidopsis thaliana suspension cell line T87 was provided by RIKEN BRC, a participant in the National Bio-Resource Project of the MEXT (Ministry of Education, Culture, Sports, Science and Technology), Japan. Protoplast isolation and transfection experiments for transient expression assays were performed as described by Hartmann et al. . 35Spro-GtMYB3 and 35Spro-GtbHLH1 vectors were used as effector vectors . An approximately 1-kb long 5′-upstream region prior to the translation start site of the torenia F3H gene was amplified and used as the TfF3H promoter. TfF3H promoters with or without transposon insertions were fused to LUC (firefly luciferase gene). The gentian GtF3H promoter was also used. 35S-RLUC (Renilla luciferase gene) was coinfected as a transformation control. Dual luciferase assays were performed as described previously .
Complementation study by transformation of torenia plants
A binary vector was constructed to express a gentian F3H cDNA (GtF3H-1 , accession no. AB193311) in CrW. The plasmid, pSKan-35SGtF3H, was transformed into Agrobacterium tumefaciens strain EHA101. Torenia transformation was performed as described previously  using kanamycin as a selection agent. The generated transgenic plants were transferred to an enclosed greenhouse and cultivated until flowering. Northern blot analysis was performed as described above using probes for torenia and gentian F3H.
Long terminal repeat
Torenia retrotransposon 1.
We gratefully thank Dr. Ryutaro Aida (National Institute of Floricultural Science, Japan) for providing us with the torenia materials and the transformation protocol. We also thank Mrs. Akiko Kubota and Mrs. Emiko Chiba, Iwate Biotechnology Research Center, for technical assistance with production of transgenic torenia plants. The work described here was financially supported by Iwate Prefecture and also in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 25660030).
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