Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen

Background Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. Results A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. Conclusion To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.


Background
Cyclamen persicum Mill. (cyclamen) is a popular and economically significant pot plant crop in Japan, Germany, Italy, the Netherlands and North America. Flower colour in commercial lines ranges from white, through red, pink, reddish-purple to purple. The pigments present are predominantly anthocyanins and there have been several studies on anthocyanin and flavonoid pigmentation in cyclamen [1][2][3][4][5]. The main anthocyanins are 3,5-di-O-glucosides of peonidin, cyanidin and malvidin ( Figure. 1). There are two missing colour groups in cyclamen, the orange-red of pelargonidin-derived anthocyanins [6] and blue, even though some delphinidin-derived anthocyanins often associated with blue flower colours are present in maroon to purple cultivars [1][2][3]6].
To date there has only been one reported molecular breeding experiment involving flavonoid pigments for cyclamen. It was focused on the generation of yellow flower colours through the production of yellow flavonoid pigments [7]. Our interest is in altering the antho-cyanin-based colours [8]. In flower colour modification studies in general, particular attention has been paid to the enzymes responsible for the hydroxylation of the Bring of the flavonoid molecule, namely F3'H and F3'5'H ( Figure. 1) because of their key influence on the colour of anthocyanin pigments [9]. Specific experiments to accumulate delphinidin-derived anthocyanins by over expression of a F3'5'H transgene have been reported for carnation [10] and rose [11], while inhibition of both the F3'H and the F3'5'H genes has been used to modify colour and promote cyanidin-and pelargonidin-based pigment accumulation in flowers in the genera Torenia [12], Nierembergia [13] and Osteospermum [14].
Our strategy for modification of flower colour in cyclamen focused on the F3'5'H. Substrate feeding experiments with DHK and the F3'H/F3'5'H inhibitor tetcyclacis indicate that the cyclamen DFR can use DHK and that cyclamen has the ability to make pelargonidinderived anthocyanins (K. Schwinn, unpublished data). The cloning of a F3'5'H cDNA and our cyclamen genetic transformation system [15] have allowed us to investigate flower colour formation in cyclamen. In this study we report on the effects of antisense suppression of F3'5'H on flavonoid end-product accumulation and flower colour.

Isolation and sequence analysis of a cyclamen flavonoid 3' , 5'-hydroxylase cDNA
A putative full-length cDNA for F3'5'H (CpF3'5'H) was isolated from a cDNA library made from mixed flower bud stages of C. persicum 'Sierra Rose'. The complete nucleotide sequence has 1719 nucleotides with a single major ORF encoding 508 amino acid residues (GenBank accession GQ891056).

Generation of transformed lines and transgene expression analyses
Antisense CpF3'5'H transformants were produced from the 'Purple' cultivar using constructs pPN48/51, and from the 'Wine-Red' cultivar using pLN96/pPN50 ( Figure. Table 1. Anthocyanin identities were assigned by retention times and mass spectrometer data and were consistent with the anthocyanins identified  previously for cyclamen, predominantly the 3-mono and 3,5 di-glucosides of peonidin, cyanidin and malvidin [2,4].  Figure 6B). Pelargonidin, an anthocyanin pigment with a monohydroxylated B-ring, was not produced in the transgenic lines of either cultivar.
There was also a marked reduction in total anthocyanin concentration in petal tissue of the transgenic lines. Lines with modified flower colour showed a decrease in total anthocyanin concentration of up to 80% of that in untransformed controls (Figure. 6B). The difference in anthocyanin concentrations between the transgenic lines and their respective controls were statistically significant at the 5% level.
Flavonol profiles were also examined. Flavonols in the untransformed and transgenic lines were putatively iden-  * Anthocyanins were identified using HPLC retention times and UV and mass spectrometer data as compared with previously published data. tified as kaempferol and quercetin 3-glucosides, rutinosides and acylated rutinosides (data not shown). This is consistent with previous studies [2]. Total flavonol concentration in the transgenic lines showed a statistically significant increase in most lines ( Table 2). The quercetin/kaempferol ratio also increased significantly in most transgenics lines of cv 'Purple' but decreased significantly in all the transgenic lines of cv 'Wine-Red' ( Table 2).  Table 3). The exceptions were lines 31675 (cv 'Purple') and 31698 (cv 'Wine-Red') for L and C values. Both lines exhibited the least change in their anthocyanin profiles (Figures 6A,  B). The majority of transgenic lines of both cultivars showed an increase in lightness (L) and a reduced intensity of colour (C). This is consistent with the decreased anthocyanin concentration in the petal tissue from the transgenic lines. There was also a clear shift in H° away from purple in the control line towards red in the transgenic lines of the 'Purple' cultivar. This change in hue angle correlates with a decrease in the proportion of delphinidin-derived anthocyanins. However line #31685, which had the largest proportion of cyanidin-derived pigments, did not have the largest shift in H°. Similarly the only line of the cv 'Wine-Red' transgenics showing a shift from delphinidin-to cyanidin-derived pigments (line #31691) did not show a significant change in H° while the other two transgenics did. The shift in hue angle for the cv 'Wine-Red' transgenics was in fact back to the purple region of the colour wheel. The overall shift, however, was very small and hue angle remained in the red/pink region.

Discussion
Antisense suppression of CpF3'5'H was successful in changing anthocyanin profiles and flower colour in cyclamen. A shift from predominantly delphinidin-derived  pigments to a greater relative proportion of cyanidinderived pigments was achieved and in general this showed up as a concomitant shift in H°, the parameter indicating colour group. It is interesting that the degree of change in H° did not correlate with the degree of shift in pigments. The fact that the transformants also showed variable drops in total anthocyanin levels and changes in flavonol level and type illustrates both the links between the different pools of flavonoid substrates and the importance of the roles that anthocyanin concentration and flavonol copigmentation play in flower colour. Similar changes in anthocyanin concentration and the accumulation of cyanidin-derived anthocyanins were seen for the two different minicyclamen cultivars and yet the greatest change in H° was seen in the lines of the purple cultivar. This is most likely due to a reduction in the predominant anthocyanin, malvidin 3-5-di-O-glucoside in these lines. This anthocyanin has been reported as being bluer in colour than malvidin mono-glucosides [3]. The predominant anthocyanin in the 'Wine-Red' cultivar is malvidin 3-O-glucoside and this has been reported to give pink/purple colours, closer to the colour associated with cyanidin and peonidin pigments [3].
Pelargonidin-based pigments were not detected in the flowers of the transgenics. One explanation for their absence is that suppression of F3'5'H activity was not complete, as evidenced by the presence of delphinidinderived anthocyanins. This may be either due to inefficiency of the antisense approach (as opposed to hairpin RNA-induced RNAi [18]), effects due to transgene insertion or copy number [19][20][21], or the presence of other unaffected F3'5'H family members. The presence of a F3'H enzyme in the petals could have also removed substrate for pelargonidin production. We have searched for a cyclamen F3'H cDNA and found one (GenBank GU808358) with high deduced amino acid similarity to known F3'H sequences of other species (81% identity with the F3'H of gentian). However, transcript levels for this particular F3'H gene were not detectable by northern analysis during cyclamen petal development (unpublished data).
Substrate specificity is an important consideration regarding pelargonidin production. In some species, such as petunia [22], cymbidium [23,24] and Osteospermum [14], synthesis of pelargonidin-based anthocyanins is limited by the substrate specificity of the endogenous DFR. Our substrate feeding experiments (mentioned previously) showed that cyclamen has the ability to make pelargonidin-derived anthocyanins. It is still possible, however, that cyclamen DFR has low substrate specificity for DHK and the action of flavonol synthase (FLS), F3'H and F3'5'H means that the DHK substrate is not used for the synthesis of pelargonidin. Retransformation of an antisense F3'5'H line from this study, with a transgene encoding a DFR known to efficiently catalyse the reduction of DHK to leucopelargonidin [25][26][27] could result in transgenic plants accumulating pelargonidin derivatives in flowers, as successfully demonstrated for Osteospermum [14]. It remains to be resolved whether there is a F3'H functioning in the flower. The presence of cyanidinbased pigments in the flowers of the antisense CpF3'5'H lines suggests F3'H activity. Thus, inhibition of either F3'H or FLS gene activity to reduce enzymatic competition for DHK substrate may also be necessary to promote pelargonidin production in DFR/antisense F3'5'H transgenics.
In the cyclamen transgenic lines, total anthocyanin levels decreased markedly while flavonol levels increased and the quercetin/kaempferol ratio changed. Similar results were reported for Nierembergia flowers modified with an antisense F3'5'H construct and were suggested to be due to a modified flow through the flavonoid pathway [13]. A block in F3'5'H activity resulted in an increase in pelargonidin precursors. Low F3'H activity coupled with a DFR that putatively does not recognise DHK, was suggested to have led to limited substrate flow toward pigment production and an increase in the sustrate pool for FLS [13]. The flavonoid enzyme kinetics are not known for cyclamen. However, if the cyclamen DFR has a low specificity for pelargonidin or cyanidin precursors (as the reduction in total anthocyanins ( Figure. 6B) suggests) this would provide extra substrate for the FLS enzyme and explain the increased flavonol levels. Competition for substrate between FLS and DFR has also been shown to occur in petunia [28,29].
It is interesting that while flavonol levels generally increased in the transgenics, there were differences in the quercetin/kaempferol ratios between the lines of the different cultivars. Quercetin flavonols increased in cv 'Purple' lines while kaempferol types increased in cv 'Wine-Red' lines. This inverse result and the consistency of the ratio change within lines of each cultivar argues against the suppression of F3'5'H activity directly altering the balance of DHK and DHQ, and thus what is available for the FLS. Furthermore, differing substrate specificities of their respective FLS cannot account for the observed results. Differing specificities of other enzymes are likely to be the cause. The probable candidate is F3'H, which in other species can not only alter the balance between DHK and DHQ, but also convert kaempferol to quercetin [30]. Further studies of cyclamen flower colour would warrant a continued search for a F3'H.

Conclusions
We report here the first successful alteration of cyclamen anthocyanin pigmentation using genetic modification techniques. Our results highlight the intricate interplay between type and concentration of both anthocyanin pig-ments and flavonol co-pigments in flower colour and illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products.

Cloning of F3'5'H cDNA and sequence analysis
A cDNA library from mixed flower stages of C. persicum 'Sierra Rose' petals was made using a lambda ZAPII bacteriophage vector kit (Stratagene, USA). This library was first screened with a heterologous clone of F3'H from petunia (Florigene Flowers, Australia) and a partial F3'5'H cDNA was found. The partial F3'5'H cDNA was used to rescreen the cDNA library to obtain a full length CpF3'5'H cDNA.
The MegAlign programme of Lasergene (DNASTAR Inc., Madison, USA) was used to compare the CpF3'5'H deduced amino acid sequence to ten known F3' 5

Transformation with Agrobacterium tumefaciens
Etiolated hypocotyls of two parental lines of F1 hybrid minicyclamen cv 'Purple' and cv 'Wine-Red' were used as explants for transformation experiments. A. tumefaciens strain EHA105 containing either pLN96, pPN48, pPN50 or pPN51 were used to inoculate explants. The transformation protocol used was that reported by Boase et al. [15] except that hygromycin was used as the selection agent for cv 'Purple' lines using a range of concentrations: 5mg/l to day 12 after Agrobacterium inoculation, 20mg/l to day 77 after inoculation, then 15mg/l until shoots were recovered.

Northern blot analyses
RNA was extracted from petal tissue for northern blot analysis using a modified hot borate method [33,34]. RNA was separated by electrophoresis on a 1% agarose RNA gel and subsequently transferred to Hybond XL nylon membranes using a SSC overnight blotting method. The membranes were hybridized with appropriate radioactively-labelled probes. The probe for hpt was a 1.1 kb XhoI fragment digested from pCAMBIA1301, which contained the hpt gene. The probe for F3'5'H was a 1.7 kb XbaI-EcoRI fragment digested from pLN95. Both membranes were also rehybrised to a cDNA probe corresponding to a 25/26S rRNA (pTip6) from Asparagus officinalis, to show RNA loadings. Autoradiography was conducted at -80°C using Kodak Biomax X-ray film.

RT-PCR analysis of nptII mRNA transcripts
To investigate the expression of the introduced nptII selectable marker recombinant gene, RT-PCR analysis was performed on RNA extracted from petals using a modified hot borate method [33,34]. Three independent transgenic lines of cv 'Wine-Red' (#31691, #31695 and #31698) and one untransformed control (#29009) were tested. First strand cDNA was reverse transcribed from 100ngRNA per sample using Superscript II (Invitrogen USA) and oligo dT primer, and then 1 μl of the resulting cDNA per line was used for the PCR. For PCR, initial denaturation was at 94°C for 2 min followed by 40 cycles of melting (94°C/30 s), annealing (50°C/30 s) and extension (72°C/2 min). The nptII primers used were: forward 5'-ATGACTGGGCACAACAGACCATCGGCTGCT-3' and reverse, 5'-CGGGTAGCCAACGCTATGTCCTGA-TAGCGG-3'.
PCR products were separated electrophoretically on a 1% (w/v) NaB agarose gel stained with Sybr ® safe (Invitrogen USA).

Flavonoid analyses
Flavonoids were analysed by high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). Freeze dried tissue was used for the analysis. Samples of ground freeze-dried petal tissue (50mg DW) were extracted initially in 2ml of methanol:acetic acid:water (70:3:27) and then reextracted in 2 ml methanol:acetic acid:water (90:1:9). The combined supernatants were concentrated in vacuo and made up to a final volume of 1ml. HPLC analysis was carried out using a Waters 600 solvent delivery system with a Phenomenex Prodigy (5 μm, 250 × 4.6 mm) RP-18 end-capped column (column temperature 30°C) and a Waters 996 PDA detector. Solvent systems, flow rates and gradients are as described by Bloor et al. [35]. Flavonoids were detected at 350nm and anthocyanins at 530nm. Flavonoid levels were determined as quercetin-3-O-rhamnoglucoside (Apin Chemicals, Abingdon, Oxon, UK) equivalents, and the anthocyanins as cyanidin 3-O-glucoside (Extrasynthese, Genay, France) equivalents. Results are reported as the mean of the two replicates.
Separate extracts were analysed by electrospray mass spectrometry with a Thermo Finnigan LTQ ion-trap mass spectrometer. A Synergi Fusion RP80, 4 μm, 150 × 2.1 mm column with 4 × 2 mm guard cartridge from Phenomenex Ltd was used for separation. The mobile phase consisted of water (A) and acetonitrile (B) both containing 1% formic acid (FA). Extracts were injected at 5 μL volumes with a gradient program from 95% A to 50% A over 50 min. The column was washed by ramping to 90% B for 5 min and then re-equilibrated to the starting conditions for a further 5 min. Compound elution was monitored by PDA detector scanning the range 250-600 nm and by mass scanning from m/z 150-1500 to collect parent, MS 2 and MS 3 data in positive and negative ion (additional run) selection modes.

Flower colorimeter analysis
Colours in all lines were quantified by measurement of three petals of each flower, three flowers per line with a Minolta CR-200 tristimulus colorimeter, set on CIELab D65 light source and 0° observer angle. Lightness (L) represents the proportion of total incident light that is reflected. Chroma (C) describes the degree to which selective absorption occurs i.e. colour saturation in relative intensity units. Hue angle (H) is derived from a CIE-LAB colour space wheel with values stepped counterclockwise from red at 0°/360°, yellow at 90°, bluish-green at 180° and blue at 270° [36].

Statistics
A one-way ANOVA was performed on each data set shown in Tables 2 &3 and Figure 6B followed by a comparison of means using either a 5% Fisher's Least Significant Difference (5% LSD) to compare each line with a single control, or contrasts to compare each line with the combined mean of two controls. Lines with values significantly different from their control (or pair of controls) at the 5% level have been indicated by adding a superscript a to the means in Tables 2 and 3, and in Figure 6B. All analyses were performed using GenStat statistical software [37].