Flavonoid biosynthesis in white-¯owered Sim
carnations (
Dianthus caryophyllus
)
Masami Mato
a,*, Takashi Onozaki
a, Yoshihiro Ozeki
b,
Daisuke Higeta
b, Yoshio Itoh
b, Yasuko Yoshimoto
b,
Hiroshi Ikeda
a, Hiroyuki Yoshida
c, Michio Shibata
aa
Department of Floriculture, National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2392, Japan
b
Department of Biotechnology, Tokyo University of Agriculture and Technology, Naka-machi, Koganei, Tokyo 184-8588, Japan
c
Japan Tobacco Inc., Applied Plant Research Laboratory, 1900 Idei, Oyama, Tochigi 323-0808, Japan
Accepted 8 November 1999
Abstract
Analysis of ¯avonoid composition and gene expression of enzymes involved in anthocyanin synthesis in ¯owers of four acyanic and one cyanic cultivar of Sim carnation showed that the acyanic ¯ower cultivars are divided into three types. The ®rst includes two normal white cultivars, `U Conn Sim' and `White Sim'; the second includes a nearly pure white cultivar, `Kaly'; and the third includes a nearly pure white cultivar, `White Mind'. `U Conn Sim' and `White Sim' accumulated ¯avonol glycosides and lacked anthocyanins. The transcription of the several genes of enzymes involved in ¯avonoid biosynthesis were reduced at a later ¯owering stage than the cyanic cultivar, especially the genes encoding dihydro¯avonol 4-reductase and anthocyanidin synthase. `Kaly' accumulated ¯avanone glycosides and a small amount of ¯avonol and ¯avone glycosides by blocking the transcription of the gene encoding ¯avanone 3-hydroxylase, in addition to the transcriptional reduction of the genes for ¯avonoid biosynthesis at a later ¯owering stage. Although `White Mind' contains little ¯avonoid, the position of the block on ¯avonoid biosynthesis in `White Mind' is not known.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Dianthus caryophyllus; White ¯ower; Flavonoid; Flavanone 3-hydroxylase; Dihydro-¯avonol 4-reductase; Anthocyanidin synthase
*Corresponding author. Present address: Akita Agricultural Experiment Station, 111 Konakasima, Nida, Akita 010-1426, Japan. Tel.:81-18-839-2121; fax:81-18-839-2359.
E-mail address: matou@mtj.biglobe.ne.jp (M. Mato).
1. Introduction
An important aim in ¯oriculture is to obtain pure white acyanic ¯owers in addition to cyanic ones. The relationship between white coloration and a block on ¯avonoid biosynthesis has been investigated inAntirrhinum majus(Harrison and Stickland, 1974; Stickland and Harrison, 1974; Forkmann and Stotz, 1981; Spribille and Forkmann, 1981), Petunia hybrida (Kho et al., 1977; Mol et al., 1983) and Matthiola incana (Forkmann et al., 1980; Heller et al., 1985). The color of most white or ivory acyanic ¯owers is caused by a de®ciency of ¯avanone 3-hydroxylase (F3H), dihydro¯avonol 4-reductase (DFR), or antho-cyanidin synthase (ANS) in ¯avonoid biosynthesis (Fig. 1). However, in
Antirrhinum majus, the pure white (albino) ¯ower of the niv mutation does not
have ¯avonoid compounds and ¯avonoid biosynthesis is blocked at the chalcone synthase (CHS) step (Fig. 1). Thus, the relationship between white coloration and the block on ¯avonoid biosynthesis varies among plant species.
It is known that in acyanic strains of Dianthus caryophyllus, having the recessive alleleaa, ¯avonoid biosynthesis is interrupted between dihydro¯avonol and ¯avan-3,4-diol by a de®ciency of DFR (Stich et al., 1992a), but the other blocks have not been determined.
About 400 bud mutants have originated from the cultivar `William Sim'; these are called Sim carnations. Our investigation used ®ve cultivars of Sim carnations: `Kaly' and `White Mind' (nearly pure white cultivars), `U Conn Sim' and `White Sim' (white), and `Scania' (red) (Fig. 2). The pedigree is shown in Fig. 3. The parents of the bud mutants, `William Sim' and `Ember Sim', were so old that they could not be obtained; therefore, `Scania' was used as the red cultivar in this research. We have already reported that these four acyanic cultivars of Sim carnations can be divided into three types based on the difference of ¯avonoid composition (Onozaki et al., 1999). The ®rst includes two white cultivars, `U Conn Sim' and `White Sim', containing ¯avonols as main ¯avonoid; the second
Fig. 2. Phenotypes of Sim carnation used in this study. `White Mind' (upper left), `U Conn Sim' (upper right), `White Sim' (lower left), `Scania' (lower center), `Kaly' (lower right).
includes a nearly pure white cultivar, `Kaly', containing ¯avanones as main ¯avonoid; and the third includes a nearly pure white cultivar, `White Mind', containing little ¯avonoid. From this result we have been estimated the blocking points in ¯avonoid biosynthesis. Further, northern analysis clari®ed the way they regulate ¯avonoid biosynthesis.
2. Materials and methods
2.1. Plant materials
The investigations were performed with ®ve cultivars of Sim carnation, `Kaly' and `White Mind' (nearly pure white cultivars), `U Conn Sim' and `White Sim' (white) and `Scania' (red) (Fig. 2).
Plants were cultivated in a greenhouse. Flower buds at the following ®ve stages were used for materials:
Stage 1. Closed flower buds, 20 mm long.
Stage 2. Just opening flower buds, 25 mm long.
Stage 3. Opening flower buds. The visible part of the petals is 1 mm long.
Stage 4. The visible part of the petals is 5 mm long.
Stage 5. The visible part of the petals is 10 mm long.
2.2. Extraction and analysis of ¯avonoids
The petals were collected and then extracted in ca. 20 ml MeOH at room temperature overnight. The MeOH extract was evaporated under reduced pressure, and the residue was dissolved in ca. 20 ml H2O. The solution was shaken in a separating funnel with petroleum ether. The aqueous phase was evaporated to dryness and the residue was dissolved in 1 ml (MeCN:H2O:H3PO4, 10:90:0.2) per gram fresh weight. The solution was ®ltered through a cellulose acetate ®lter (0.45mm pore size, Dismic-13 cp), and 5ml was applied to a C18 reversed-phase column (LiChrospher 100 RP-18) in a high performance liquid chromatography (HPLC) with JMBS DP-L 915W by the linear gradient solvent system (MeCN:H2O:H3PO4 grading from 10:90:0.2 to 30:70:0.2), at a ¯ow rate of 1 ml minÿ1. The absorbance at the maximum wavelength of each peak in the HPLC elution of the water phase was monitored, and the ¯avonoid content of petals was determined by peak area.
2.3. Extraction and analysis of anthocyanin
UV±Vis recording spectrophotometer UV-240 at 530 nm to determine the anthocyanin content.
2.4. Isolation of RNA
Total RNAs were prepared from the petals of a bud. The sample tissues were pulverized in liquid N2, and dissolved in 20 ml of emulsion of extraction buffer, 100 mM Tris±HCl, pH 9.0, 300 mM NaCl, 10 mM EDTA, 14 mM 2-mercaptoethanol:100 mM Tris±HCl, pH 9.0, saturated PhOH (1:1); then 10 ml of Seavag's mixture (CHCl3:isoamyl alcohol, 24:1) was added and mixed just before centrifugation at 10 000g. Protein was extracted three times with 20 ml of PhOH:Seavag's mixture (1:1) and once with 10 ml of CHC13. After polysacchar-ides were removed by the addition of 300 mM NaOAc on ice for 20 min and centrifugation at 10 000g for 20 min, the nucleic acids were precipitated by the addition of 2.5 volumes of EtOH. The precipitates were dissolved in TE (10 mM Tris±HCl, pH 7.5, and 1 mM EDTA) containing 10 units mlÿ1
of RNase inhibitor from human placenta and 1 mM dithiothreitol. The RNAs were precipitated by the addition of LiCl (2 M ®nal concentration) and then dissolved in the same buffer, layered on a 5.7 M CsCl cushion, and precipitated by centrifugation at 145 800gfor 16 h (Mato et al., 1998). Poly(A)
RNAs were isolated from 1 mg of total RNAs by two cycles of oligo(dT)-latex af®nity chromatography (OligoTM-dT3O ``Super''). A cDNA library was constructed with a l ZAPII-cDNA/Gigapack III Gold cloning kit from 1mg poly(A)
RNA prepared from young petals of cyanic ¯ower buds as described by the manufacturer, except for additional reaction with AMV reverse transcriptase after reverse transcription by the manufacturer's methods.
2.5. cDNA cloning
The cDNA clones of PAL, CHS and DFR were obtained by plaque hybridization to the library using carrot PAL cDNA (Takeda et al., 1997), CHS cDNA (Ozeki et al., 1993) and Japanese morning glory DFR cDNA (Inagaki et al., 1994) as probes labeled by DIG-High Prime. Prehybridization and hybridization were done in a solution of 5SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, and 1% blocking reagent at 558C. After hybridization, the membranes were washed twice for 10 min at room temperature in 2SSC, 0.5% SDS, then twice for 10 min in 1SSC, 0.1% SDS, and ®nally twice for 30 min at 458C in 1SSC, 0.1% SDS. A DIG-DNA labeling and detection kit was used for immunological detection by the manufacturer's methods, and the sheets were exposed onto X-ray ®lm.
of TTG-TTA-GGG-ACG-AGG-ATG-AAC-GTC and GCC-AAT-GGG-TAG-ACC-GTC-GCG-TCT for F3H (X72592) (Britsch et al., 1993); and CAG-GTC-CCG-ACT-ATA-GAC-CTC-AAG and TCC-TTC-GGC-GGT-TCA-CAG-AAA-ACT for ANS (Henkel and Forkmann, U82432). The cDNA sequences of double strands were determined by the dideoxynucleotide chain termination method (Sanger et al., 1977) with a Thermo SequenaseTM cycle sequencing kit and IRD 41 primers of T3, T7, M13 forward, or M13 reverse using a LI-COR DNA sequencer model 4000.
2.6. RNA blot analysis
Electrophoresis of 1mg of poly(A)
RNA was done in a denatured formaldehyde±agarose gel and blotted onto a Nytran-plus membrane ®lter. As probes, cDNA clones were labeled with DIG-High Prime and used for northern hybridization analysis. Prehybridization and hybridization of the membranes were done in a solution of 5SSC, 50% deionized formamide, 0.1% N -lauroylsarcosine, 0.02% SDS, and 2% blocking reagent at 4208C overnight; the probe was added for the hybridization step. The membranes were washed twice for 10 min at room temperature in 2SSC, 0.1% SDS, twice for 10 min in 0.1SSC, 0.1% SDS, and ®nally twice for 30 min at 6808C in 0.1SSC, 0.1% SDS. A DIG-DNA detection kit was used for immunological detection according to the manufacturer's instructions. The sheets were exposed onto X-ray ®lm.
2.7. RT-PCR analysis experiment
RT-PCR was done with an RNA PCR kit (AMV Version 2.1) according to the manufacturer's protocol, after DNase (RNase-free) treatment at 2508C for 15 min. The cDNAs for F3H, DFR and ANS were ampli®ed from poly(A)
RNA as a template for RT-PCR using primer sets of ATG-GTC-GCT-GAA-AAA-CCC-AAA-ACG and CTA-AGC-AAG-TAT-TTG-GTC-AAT-AGA for F3H; ACA-TAG-TTT-AGT-TTA-AGC-TCG-GTA and TTA-TTT-AAA-AAA-TAT-AAG-CGT-CAC for DFR; and GAA-TTC-CAC-GAA-AAT-CGC-TCC-GTC and TCA-CTG-GGC-ATT-GGA-CAT-CCT-GAG for ANS.
3. Results
3.1. Gene expression of white-¯owered cultivars `U Conn Sim' and `White Sim'
classes. The ®rst includes two white cultivars, `U Conn Sim' and `White Sim', and one red cultivar, `Scania'; the second includes a nearly pure white cultivar, `Kaly'; and the third includes a nearly pure white cultivar, `White Mind'. The second and third classes are discussed in Section 3.2.
The ®rst class had 10 peaks atRt2.1, 3.3, 3.6, 5.5, 19.4, 21.2, 23.1, 27.1, 29.8 and 30.4 min (1±10, Fig. 4a). Peaks 5 and 6 agreed with retention times and absorption maxima of the ¯avonol (kaempferol) glycosides that had been identi®ed by Onozaki et al. (1999). The other peaks are assumed to be organic acids, having maximum wavelengths of 245±265 nm.
`U Conn Sim', `White Sim' and `Scania' contain mainly ¯avonol glycosides, and `Scania' contains anthocyanins. Therefore, HPLC analysis of the ¯avonol glycoside content was done for each of the ®ve ¯ower bud stages in these two
cultivars and `Scania' (Fig. 5a). The ¯avonol glycoside content was high at stage 1 and then gradually decreased in all three cultivars. Anthocyanin content of `Scania' was also analyzed spectrophotometrica (Fig. 5b); the content was almost zero at stage 1 and increased as the ¯avonol content decreased. White ¯owers generally contain ¯avone and ¯avonol glycosides for ¯ower pigments (Reznik, 1956), and anthocyanins are present in cyanic ¯owers (Harborne, 1967; Timberlake and Bridle, 1975).
We used northern blot analysis at the 5-¯ower-bud stages to measure mRNA expression levels in ¯avonoid biosynthesis by `U Conn Sim', `White Sim' and `Scania' (Fig. 6) because of a time lag between ¯avonoid and anthocyanin
Fig. 5. Changing patterns of (a) ¯avonols and (b) anthocyanins. The values of ¯avonols are expressed as percentage of total area of peaks 5 and 6 in Fig. 4a. The values of anthocyanins are expressed as percentage of the absorbance in `Scania' at stage 5 recorded at 530 nm. (&) `U Conn Sim'; (D) `White Sim'; (*) `Scania'.
biosynthesis (Fig. 5). Northern blot analysis was done using cloned cDNAs of phenylalanine ammonia lyase (PAL) (ABXXXXXX, unpublished until the ®rst proof), CHS, F3H, DFR, ANS, and actin (load control) as probes (Fig. 6). The mRNA expressions of PAL and actin were high in all three cultivars at all stages. The mRNA expressions of CHS and F3H were high until the later stages in `Scania' (lane 3), and high in `U Conn Sim' (lane 1) and `White Sim' (lane 2) until stage 2. DFR and ANS mRNAs were not detected in `Scania' until stage 2, and were not detected at any stage in `U Conn Sim' and `White Sim'. RT-PCR analysis at stage 4 was used to determine whether the transcription of DFR and ANS was a reduction or a defect (Fig. 7). The cDNAs from the mRNAs of DFR and ANS at stage 4 were detected in `U Conn Sim' (lane 1) and `White Sim' (lane 2) by RT-PCR; this result seemed to be showing a transcriptional reduction of the genes encoding DFR and ANS. We assume that the defects in the anthocyanin of `U Conn Sim' and `White Sim' are the results of the transcriptional reduction of the genes encoding CHS, F3H, DFR and ANS at a later stage than in `Scania', especially because of limited expression of DFR and ANS at all stages.
3.2. Gene expression in nearly pure white cultivars `Kaly' and `White Mind'
In `Kaly', ¯avonol glycosides (peaks 5 and 6) were lower than in `U Conn Sim', `White Sim' and `Scania' (Fig. 4b). `Kaly' had peaks atRt16.1, 17.2, 27.7 and 34.7 min (11±14, Fig. 4b). Peak 13 agreed with ¯avanone (naringenin) glycosides that had been identi®ed by Onozaki et al. (1999) and the hydrolysate of peak 14 was also identi®ed as naringenin. Peaks 11 and 12 are assumed to be ¯avone glycosides, having maxima at wavelengths of 270 and 335 nm, but the amount of peaks 11 and 12 were too small to allow us to identify the hydrolysates. In `White Mind', ¯avonoid compounds indicated by peaks 5, 6, 11, 12, 13 and 14 were present in trace quantities, and only organic acids seemed to accumulate (1, 2, 3, 4, 7, 8, 9, 10, Fig. 4c).
The two nearly pure white cultivars differed from the two white ones in ¯avonoid composition; so we used northern blot analysis of `Kaly', `White Mind', the two white cultivars, and `Scania' to determine the mRNA levels during ¯avonoid biosynthesis in the two nearly pure white cultivars (Fig. 8). mRNAs were prepared at bud stages 1 and 4 for northern blot analysis because of the separation of the mRNA expression level during ¯avonoid biosynthesis into early
and late stages (Fig. 6). Northern blot analysis was done by using cDNAs of PAL, CHS, F3H, DFR and ANS as probes (Fig. 8). mRNAs of F3H, DFR and ANS were not detected in `Kaly' at stage 1 or 4 (lane 5). RT-PCR analysis at stages 1 and 4 was used to determine whether the transcription of F3H was a reduction or a defect (Fig. 9). The cDNA from the mRNA of F3H at stages 1 and 4 was not detected by RT-PCR, but DFR and ANS were detected at stage 4 (lane 5). This result seemed to be showing a transcriptional block on the genes encoding F3H. The defects in anthocyanin and the reduction of the ¯avonol glycosides in `Kaly' (lane 5) seem to be due to a block on the transcription of the F3H gene at all stages accompanied by the transcriptional reduction of CHS, DFR and ANS at later stages, as in the white cultivars (lanes 2 and 3). On the other hand, the expression of CHS and F3H mRNAs was reduced at stage 4, and the mRNAs of DFR and ANS were not detected in `White Mind' (lane 1), as in the white cultivars. The reason why few ¯avonoids accumulated in `White Mind' could not be determined from this northern blot analysis. Moreover, although we also measured the activities of hydroxycinnamate:CoA ligase (4CL), CHS and chalcone isomerase (CHI) in crude extracts, activity of all these enzymes was found in `White Mind' (data not shown). The reason why few ¯avonoids accumulated in `White Mind' could not be determined from these enzyme assays. Because `White Mind' seemed to accumulate little ¯avonoid (Fig. 4c), we are sure that this is blocked before ¯avonoid biosynthesis.
Fig. 8. Northern blots showing hybridization of PAL, CHS, F3H, DFR, ANS and actin cDNA probes at bud stages 1 and 4. Lane 1: `White Mind'; 2: `U Conn Sim'; 3: `White Sim'; 4: `Scania'; 5: `Kaly'.
4. Discussion
All ®ve cultivars Ð `Kaly', `White Mind', `U Conn Sim', `White Sim' and `Scania' are bud mutants that originate from `William Sim' (Fig. 3). Northern blot (Figs. 6 and 8) and RT-PCR analysis (Figs. 7 and 9) showed that the transcription of the genes encoding CHS, F3H, DFR and ANS in `Kaly', `White Mind', `U Conn Sim' and `White Sim' seem to be reduced at later bud stages than in `Scania'. The data suggests two phases of ¯avonoid metabolism; one early in bud development where ¯avones/¯avonols are made, and a later phase where anthocyanins are made following the induction of DFR and ANS. This would then suggest a regulatory system that activates CHS, F3H, DFR and ANS later in development, a system that is defective in white varieties. The same system might also activate CHS and F3H transcription levels later in development, although in the white varieties a reduced level of these seem to involve quantitative regulatory systems rather than absolute blocks. In white cultivars, it seems that the accumulation of ¯avonol glycosides and the lack of anthocyanin is due to the functional expression of the genes encoding CHS and F3H in the early stages and the transcriptional reduction of the genes for CHS, F3H, DFR and ANS in the later stages, and especially DFR and ANS at all stages. This reduction and the delay in anthocyanin biosynthesis compared with ¯avonol biosynthesis in `Scania' (Fig. 5) suggests that ¯avonol and anthocyanin biosynthesis might be regulated by different transcriptional means. It has been reported that, in cultivar `Tanga', the activities of CHS and F3H, involved in the biosynthesis of ¯avonol and anthocyanin, and of ¯avonol synthase (FLS), involved in ¯avonol biosynthesis, are high in the early stages, and then DFR activity, involved in anthocyanin biosynthesis, increases in the later stages (Stich et al., 1992a,b). The separate regulation of ¯avonoid and anthocyanin biosynthesis has been documented from the relation to evolution by Koes et al. (1994). It is likely from these reports that the regulation of the biosynthesis of ¯avonol and anthocyanin is different too.
proto-oncogenes and myc-like genes, respectively, which bind directly to the promoter region of the responsive structural genes to activate their expression (Paz-Ares et al., 1987; Dellaporta et al., 1988; Cone and Burr, 1989; Perrot and Cone, 1989; Roth et al., 1991; Tonelli et al., 1991; Goff et al., 1991, 1992). In our experiment, we also observed reductions in CHS, F3H, DFR, and ANS mRNAs in white and nearly pure white Sim carnations, compared with `Scania' (similar to wild-type). It seems that the transcriptional activation factor that regulates the structural ¯avonoid biosynthetic genes has been deleted in the white and nearly pure white Sim carnations.
From the northern blot (Fig. 8) and ¯avonoid analyses (Fig. 4), transcription of the F3H gene in `Kaly' seems to be blocked at all stages, accompanied by the transcriptional reduction of CHS, DFR and ANS in the later stage, as in the white cultivars. It has been reported that transcription of the F3H gene on a white background is blocked in `Aladin', which has ¯owers showing thin red stripes on a white background (Dedio et al., 1995). `Kaly' was derived from `Ember Sim' (Fig. 3), which has red ¯owers, like `Scania'. We estimate that the spontaneous mutation of a regulator gene in `Kaly' concerned with transcription at the later stages occurred, accompanied with an F3H gene mutation in `Ember Sim'. It might be con®rmed by progeny analysis between `Kaly' and `Aladin' whether `Kaly' carries an F3H mutation.
Although an early step in ¯avonoid biosynthesis may be blocked in `White Mind', as in the pure white (albino) ¯ower of the niv mutation of Antirrhinum majus (Martin and Gerats, 1992), neither the northern blot analysis (Fig. 8) nor the enzyme assays (data not shown) could explain why little ¯avonoid accumulates in `White Mind'.
Most mutants of the genes regulating white ¯ower color are recessive, butEluta
is a semi-dominant gene that restricts the ¯ower pigmentation to the central region of the face, the inner edges of the back lobes, and the base of the corolla tube (Martin et al., 1991). Our results cannot show which regulatory-like genes in Dianthus caryophyllus are recessive or dominant; we are now doing crosses with `Kaly', `White Mind', `U Conn Sim', `White Sim' and other cultivars to clarify this. To de®ne the regulation mechanism, it will be necessary to analyze the structure of genes and the transcription factors associated with ¯avonoid biosynthesis.
Acknowledgements
We wish to thank Dr. Masaatsu Yamaguchi for providingp-coumaroyl-CoA.
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