Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue3-4.June2000:
Scientia Horticulturae 84 (2000) 333±347
Flavonoid biosynthesis in white-¯owered Sim
carnations (Dianthus caryophyllus)
Masami Matoa,*, Takashi Onozakia, Yoshihiro Ozekib,
Daisuke Higetab, Yoshio Itohb, Yasuko Yoshimotob,
Hiroshi Ikedaa, Hiroyuki Yoshidac, Michio Shibataa
a
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: [email protected] (M. Mato).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 4 0 - 5
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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 in Antirrhinum 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 anthocyanidin synthase (ANS) in ¯avonoid biosynthesis (Fig. 1). However, in
Antirrhinum majus, the pure white (albino) ¯ower of the niv mutation does not
Fig. 1. Pathway for synthesis of ¯avonoids showing the enzymatic steps and the metabolic role of
PAL in the supply of 4-coumaroyl-CoA precursors for ¯avonoid biosynthesis (Terahara and
Yamaguchi, 1986).
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
335
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).
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 allele aa, ¯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. 3. Pedigree of the Sim carnations used in this study.
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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 (0.45 mm pore size, Dismic-13 cp), and 5 ml 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
The petals were collected and extracted in 10 ml MeOH (1% HCl) per gram
fresh weight at room temperature overnight. After removal of the insoluble
materials with ®lter paper, the absorbance of the extract was measured with a
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
337
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 2mercaptoethanol: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 polysaccharides 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 800g for 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 ZAPIIcDNA/Gigapack III Gold cloning kit from 1 mg 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 5 SSC, 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 2 SSC, 0.5% SDS, then twice for
10 min in 1 SSC, 0.1% SDS, and ®nally twice for 30 min at 458C in 1 SSC,
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.
cDNAs for F3H and ANS were ampli®ed from l DNA prepared from the
ampli®ed cDNA library described above as a template for PCR using primer sets
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
of TTG-TTA-GGG-ACG-AGG-ATG-AAC-GTC and GCC-AAT-GGG-TAGACC-GTC-GCG-TCT for F3H (X72592) (Britsch et al., 1993); and CAGGTC-CCG-ACT-ATA-GAC-CTC-AAG and TCC-TTC-GGC-GGT-TCA-CAGAAA-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 1 mg 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 5 SSC, 50% deionized formamide, 0.1% Nlauroylsarcosine, 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 2 SSC, 0.1% SDS, twice for 10 min in
0.1 SSC, 0.1% SDS, and ®nally twice for 30 min at 6808C in 0.1 SSC, 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-CCCAAA-ACG and CTA-AGC-AAG-TAT-TTG-GTC-AAT-AGA for F3H; ACATAG-TTT-AGT-TTA-AGC-TCG-GTA and TTA-TTT-AAA-AAA-TAT-AAGCGT-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'
All stage petals (see Experimental for de®nition) were analyzed for ¯avonoid
composition by HPLC. Among all the stages, Stage 1 had the largest amount of
¯avonoid as shown in Fig. 4. The chromatogram patterns were divided into three
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
339
Fig. 4. HPLC elution pro®les of the extract from petals. (a) `U Conn Sim'; (b) `Kaly'; (c) `White
Mind'. 5 and 6: Kaempferol glycoside; 13 and 14: Naringenin glycoside. Each peak on
chromatogram used the wavelength of maximum absorbance.
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 at Rt 2.1, 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
340
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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'.
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. 6. Northern blots showing hybridization of PAL, CHS, F3H, DFR, ANS and actin cDNA
probes over time (stages 1±5). Lane 1: `U Conn Sim'; 2: `White Sim'; 3: `Scania'.
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
341
Fig. 7. RT-PCR of DFR and ANS at bud stage 4. Lane 1: `U Conn Sim'; 2: `White Sim' 3: `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'. RTPCR 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 at Rt 16.1, 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
342
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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'.
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. 9. RT-PCR of F3H, DFR and ANS. Lane 1: `White Mind'; 2: `U Conn Sim'; 3: `White Sim';
4: `Scania'; 5: `Kaly'.
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
343
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.
In mutants of Petunia hybrida at loci An1, An2 and An11, reductions were
observed in the mRNAs of PAL, CHS, CHI, F3H, DFR, UDP-glucose: ¯avonoid
glucosyltransferase (UFGT), UDP-rhamnose: anthocyanidin 3-O-glucoside
rhamnosyltransferase (RT), and anthocyanin methyltransferase when compared
with wild-type plants (Quattrocchio et al., 1993; Quattrocchio, 1994; Huits et al.,
1994). Regulatory genes Del, Eluta and Rosea of Antirrhinum majus regulate
mainly structural anthocyanin biosynthetic genes encoding F3H, DFR, ANS
and UFGT (Almeida et al., 1989; Martin et al., 1991; Jackson et al., 1992). The
greatest understanding of the action of the regulatory genes that control
expression of the structural genes of the anthocyanin biosynthetic pathway
lies in maize (Cone et al., 1986; Coe et al., 1988; Chandler et al., 1989; Ludwing
et al., 1989). The genes C1 or P1 and R or B share homology with the myb
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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, but Eluta
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 providing p-coumaroyl-CoA.
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Flavonoid biosynthesis in white-¯owered Sim
carnations (Dianthus caryophyllus)
Masami Matoa,*, Takashi Onozakia, Yoshihiro Ozekib,
Daisuke Higetab, Yoshio Itohb, Yasuko Yoshimotob,
Hiroshi Ikedaa, Hiroyuki Yoshidac, Michio Shibataa
a
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: [email protected] (M. Mato).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 4 0 - 5
334
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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 in Antirrhinum 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 anthocyanidin synthase (ANS) in ¯avonoid biosynthesis (Fig. 1). However, in
Antirrhinum majus, the pure white (albino) ¯ower of the niv mutation does not
Fig. 1. Pathway for synthesis of ¯avonoids showing the enzymatic steps and the metabolic role of
PAL in the supply of 4-coumaroyl-CoA precursors for ¯avonoid biosynthesis (Terahara and
Yamaguchi, 1986).
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
335
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).
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 allele aa, ¯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. 3. Pedigree of the Sim carnations used in this study.
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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 (0.45 mm pore size, Dismic-13 cp), and 5 ml 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
The petals were collected and extracted in 10 ml MeOH (1% HCl) per gram
fresh weight at room temperature overnight. After removal of the insoluble
materials with ®lter paper, the absorbance of the extract was measured with a
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
337
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 2mercaptoethanol: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 polysaccharides 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 800g for 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 ZAPIIcDNA/Gigapack III Gold cloning kit from 1 mg 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 5 SSC, 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 2 SSC, 0.5% SDS, then twice for
10 min in 1 SSC, 0.1% SDS, and ®nally twice for 30 min at 458C in 1 SSC,
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.
cDNAs for F3H and ANS were ampli®ed from l DNA prepared from the
ampli®ed cDNA library described above as a template for PCR using primer sets
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of TTG-TTA-GGG-ACG-AGG-ATG-AAC-GTC and GCC-AAT-GGG-TAGACC-GTC-GCG-TCT for F3H (X72592) (Britsch et al., 1993); and CAGGTC-CCG-ACT-ATA-GAC-CTC-AAG and TCC-TTC-GGC-GGT-TCA-CAGAAA-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 1 mg 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 5 SSC, 50% deionized formamide, 0.1% Nlauroylsarcosine, 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 2 SSC, 0.1% SDS, twice for 10 min in
0.1 SSC, 0.1% SDS, and ®nally twice for 30 min at 6808C in 0.1 SSC, 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-CCCAAA-ACG and CTA-AGC-AAG-TAT-TTG-GTC-AAT-AGA for F3H; ACATAG-TTT-AGT-TTA-AGC-TCG-GTA and TTA-TTT-AAA-AAA-TAT-AAGCGT-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'
All stage petals (see Experimental for de®nition) were analyzed for ¯avonoid
composition by HPLC. Among all the stages, Stage 1 had the largest amount of
¯avonoid as shown in Fig. 4. The chromatogram patterns were divided into three
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
339
Fig. 4. HPLC elution pro®les of the extract from petals. (a) `U Conn Sim'; (b) `Kaly'; (c) `White
Mind'. 5 and 6: Kaempferol glycoside; 13 and 14: Naringenin glycoside. Each peak on
chromatogram used the wavelength of maximum absorbance.
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 at Rt 2.1, 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
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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'.
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. 6. Northern blots showing hybridization of PAL, CHS, F3H, DFR, ANS and actin cDNA
probes over time (stages 1±5). Lane 1: `U Conn Sim'; 2: `White Sim'; 3: `Scania'.
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
341
Fig. 7. RT-PCR of DFR and ANS at bud stage 4. Lane 1: `U Conn Sim'; 2: `White Sim' 3: `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'. RTPCR 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 at Rt 16.1, 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
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M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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'.
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. 9. RT-PCR of F3H, DFR and ANS. Lane 1: `White Mind'; 2: `U Conn Sim'; 3: `White Sim';
4: `Scania'; 5: `Kaly'.
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
343
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.
In mutants of Petunia hybrida at loci An1, An2 and An11, reductions were
observed in the mRNAs of PAL, CHS, CHI, F3H, DFR, UDP-glucose: ¯avonoid
glucosyltransferase (UFGT), UDP-rhamnose: anthocyanidin 3-O-glucoside
rhamnosyltransferase (RT), and anthocyanin methyltransferase when compared
with wild-type plants (Quattrocchio et al., 1993; Quattrocchio, 1994; Huits et al.,
1994). Regulatory genes Del, Eluta and Rosea of Antirrhinum majus regulate
mainly structural anthocyanin biosynthetic genes encoding F3H, DFR, ANS
and UFGT (Almeida et al., 1989; Martin et al., 1991; Jackson et al., 1992). The
greatest understanding of the action of the regulatory genes that control
expression of the structural genes of the anthocyanin biosynthetic pathway
lies in maize (Cone et al., 1986; Coe et al., 1988; Chandler et al., 1989; Ludwing
et al., 1989). The genes C1 or P1 and R or B share homology with the myb
344
M. Mato et al. / Scientia Horticulturae 84 (2000) 333±347
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, but Eluta
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 providing p-coumaroyl-CoA.
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