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RESEARCH ARTICLE
© 2001 Nature Publishing Group http://biotech.nature.com
Overexpression of petunia chalcone isomerase in
tomato results in fruit containing increased
levels of flavonols
Shelagh R. Muir1, Geoff J. Collins1, Susan Robinson1, Stephen Hughes2, Arnaud Bovy3, C.H. Ric De Vos3,
Arjen J. van Tunen3, and Martine E. Verhoeyen1*
Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective
against prostate cancer. They also contain small amounts of flavonoids in their peel (∼5–10 mg/kg fresh
weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very
potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is
correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in
the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential
health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding
chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel
flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed
between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and
demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit
flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high
levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols
present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials
for tomato-based functional food products.
tive products10. In the approach detailed in this paper, heterologous
overexpression in tomato of a gene from Petunia encoding chalcone
isomerase, an enzyme involved in flavonol biosynthesis (Fig. 1), has
been used to produce transgenic fruit with an increase in peel
flavonols of up to 78 fold, mainly due to accumulation of quercetin
glycosides.
There is considerable interest in the development of food products
from plants rich in protective vitamins or other compounds with
potential health benefits. Several groups have already shown that
genetic manipulation can be used to produce plants with
improved nutritional traits; examples are Arabidopsis seeds with
elevated vitamin E levels1 and provitamin A-enriched rice and
tomato2,3.
Flavonoids are a group of plant secondary metabolites thought
to possess health-promoting properties. They occur naturally in
fruits, vegetables, nuts, seeds, and flowers, and therefore form an
integral part of the human diet. Flavonoids have been shown to
exhibit a wide range of biological activities in animal cell culture
and in vitro systems, including antioxidant and vasodilatory
actions4. Results from several epidemiological studies suggest that
increased consumption of flavonoids, as part of a balanced diet,
may help to protect against cardiovascular disease5–7. However, the
in vitro evidence for cardiovascular protection is particularly
strong for one group of flavonoids, the flavonols (e.g., quercetin
and kaempferol), making these compounds attractive targets for
nutritional enhancement of food crops.
The flavonoid biosynthetic pathway (Fig. 1) and its regulation
have been the subject of many studies8. Commercial research in this
area has thus far been directed mainly toward altering pigmentary
flavonoids known as anthocyanins, which play an important role in
flower and leaf color9. However, recent advances concerning pathway
regulation and the biochemistry of specific enzymes and enzyme
complexes have opened up several potential pathway modification
strategies to enhance the plant’s ability to synthesize flavonoid bioac-
1Unilever
470
Results and discussion
Flavonoid biosynthesis during tomato fruit development.
Nonhydrolyzed extracts of red tomato fruit peel contained naringenin chalcone (narichalcone), in addition to the flavonol glycosides
rutin (quercetin 3-rutinoside), kaempferol 3-rutinoside, and small
amounts of a quercetin 3-trisaccharide (A.L. Davis, personal communication). Aglycones of quercetin, kaempferol, and naringenin
were not detectable. Fruit flesh did not accumulate significant
amounts of flavonoids; that is, rutin was present only at the limit of
detection (0.5 mg/kg fresh weight tomato).
Figure 2 shows the development-dependent accumulation of
rutin and naringenin chalcone in fruit peel. Levels of rutin
increased during ripening. Kaempferol rutinoside was present in
small amounts (<10% of the amount of rutin) during all stages of
development except green fruit (data not shown). Naringenin chalcone was absent in green peel but increased sharply during coloring of the fruit, reaching levels of ∼1% on a dry weight basis in
turning peel before declining through the red and overripe stages.
Naringenin chalcone represents an intermediate in the biosynthesis
of flavonols and is converted into the next compound in the pathway, naringenin, by the enzyme chalcone isomerase (CHI).
Research, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK. 2Department of Biology, University of Exeter, Exeter, EX4 4QG, UK. 3Plant Research
International, PO BOX 16, 6700AA Wageningen, The Netherlands. *Corresponding author ([email protected]).
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RESEARCH ARTICLE
Initial screening of transgenic fruit involved hydrolyzing
flavonoids to their respective aglycones before analysis by HPLC. The
hydrolysis procedure also caused the nonenzymatic conversion of
naringenin chalcone into naringenin11.
Mean levels of up to 16.52 mg quercetin and 2.05 mg kaempferol
per gram dry weight were observed in hydrolyzed peel extracts of
fruit transformed with pBBC50, representing increases of 66 and
57 fold over control, respectively (Fig. 4A, B). There was a direct relationship between quercetin and kaempferol levels in hydrolyzed
extracts of transformed fruit, with those plants displaying a highquercetin phenotype also possessing high levels of kaempferol (an
approximate quercetin to kaempferol ratio of 10:1). Naringenin
chalcone levels were severely depleted in high-flavonol transformants (Fig. 4C). Given that naringenin chalcone is a substrate for
CHI, this observation suggests an increased level of CHI enzyme
activity in pBBC50 transgenics. Total flavonoids did not accumulate
above control levels in the fruit flesh or leaves of any of the pBBC50
transformants tested (data not shown).
Nonhydrolyzed extracts were analyzed to determine the form in
which flavonoids accumulated in the peel of pBBC50-transformed
plants. Figure 5A and B show HPLC chromatograms obtained
with nonhydrolyzed peel extracts from control and pBBC50transformed tomatoes, respectively. Rutin, with a retention time
(RT) of 16.8 min, represented the major quercetin glycoside in
peel of control and pBBC50-transformed tomatoes. In addition,
significant amounts of isoquercitrin (quercetin 3-glucoside)
(RT = 17.4 min) also accumulated in the peel of pBBC50transformed plants, which might suggest that a rhamnosyl
transferase has become rate limiting in the production of rutin in
highly expressing pBBC50 lines. Several peaks that were not
detectable in controls were also found in pBBC50-transformed
peel chromatograms. The peak at RT = 19.7 min appeared to contain a mixture of two compounds; the RT and absorbance spectra
of the major component corresponded to that of kaempferol
3-rutinoside, whereas that of the minor component had an
absorbance spectrum comparable to that of a quercetin glycoside.
The small peak at 20.3 min had an absorbance spectrum comparable to kaempferol 3-rutinoside, but its higher RT value indicates a
yet unknown kaempferol glycoside.
Northern analysis revealed low levels of chi transcript in peel at
all stages of development in the fruit of high-flavonol transformants (data not shown), in contrast to control tomatoes, for
which no chi transcript could be detected. Further work is
required to ascertain the exact mechanism by which upregulating
CHI messenger RNA (mRNA) leads to increases in flavonoid end
products in peel. CHI enzyme activity could represent the sole
rate-limiting step. Alternatively, increases in CHI mRNA could
bring about changes in activity of other enzymes in the pathway,
Figure 1. Flavonoid biosynthetic pathway. Included are the structures of
the major flavonol glycoside in tomato, rutin, and its precursor,
isoquercitrin.
Northern analysis of expression of the endogenous flavonoid
biosynthetic genes chalcone synthase (chs), chi, and flavonol synthase (fls) with heterologous Petunia probes revealed that both chs
and fls transcripts were detectable in peel in all developmental
stages tested, with levels peaking during the breaker and turning
stages before subsequently decreasing in the red stage (Fig. 3). The
chi transcript level was below the limit of detection in peel of all
developmental stages. This, together with the relatively high and
specific accumulation of naringenin chalcone in turning peel, suggested that one of the rate-limiting steps in flavonol biosynthesis
in peel could lie at the level of chi gene expression. Transcripts of
the chs, chi. and fls genes were undetectable in fruit flesh, in agreement with high-performance liquid chromatography (HPLC) data,
which only indicated the presence of flavonoids at the limits of
detection in this tissue. Transcripts of the chs, chi, and fls genes
were all present in young leaves.
Transformation of tomato with the chi gene from Petunia and
analysis of transgenic plants. Tomato was transformed with a binary
vector (pBBC50) containing the Petunia hybrida chi gene under the
control of the constitutive cauliflower mosaic virus (CaMV) double
35S promoter. Southern hybridization confirmed that all transgenic
plants tested contained either one or two copies of the pBBC50
transgene (data not shown).
Figure 2. Accumulation of rutin (dashed line) and naringenin chalcone
(solid line) in developing tomato peel. Flavonoids were extracted under
nonhydrolyzing conditions and analyzed by HPLC. The terms green,
breaker, turning, red, and overripe refer to fruit color and represent stages at
approximately 7, 28, 31, 39, 45, and 49 days post-anthesis, respectively.
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RESEARCH ARTICLE
A
© 2001 Nature Publishing Group http://biotech.nature.com
A
B
B
C
C
Figure 4. Flavonoid levels in the peel of transgenic tomato fruit.
Flavonoids were extracted under hydrolyzing conditions and analyzed by
HPLC. The hydrolysis procedure resulted in the chemical conversion of
naringenin chalcone into naringenin. (A) Quercetin levels. (B) Kaempferol
levels. Each bar represents the mean of two independent fruit readings
from one transgenic plant. Black bars represent plant lines transformed
with the control plasmid pSJ89. Unshaded bars represent plant lines
transformed with pBBC50. (C) Relationship between quercetin and
naringenin chalcone levels in peel. Each bar represents the mean of two
independent fruit readings from one transgenic plant. Numbers prefixed
with a G represent plants transformed with the control plasmid pSJ89.
Numbers prefixed with a C represent plants transformed with pBBC50.
The order in which plant line numbers appear does not correlate with
parts A and B.
Figure 3. Northern analysis of developing tomato fruit. Fruits were
harvested at different developmental stages, denoted as follows: green
(G), breaker (B), turning (T), and red (R), and separated into peel and
flesh. Young leaves were harvested from mature tomato plants. RNA was
separated on formaldehyde–agarose gels, blotted, and hybridized with
Petunia (A) chs-a (Β) chi, and (C) fls probes.
perhaps by feedback/forward regulation of mRNA or protein
expression, or by stabilization of biosynthetic complexes containing CHI (ref. 12).
Transcripts of chi were present at relatively high levels in leaf samples and in green, breaker, and turning flesh from pBBC50 transgenic
plants (data not shown).
Stability and phenotype of pBBC50 transgenic plants. A number
of single-insert pBBC50 transgenic lines were selected for further
evaluation over several subsequent generations. The high-flavonol
phenotype was shown to segregate with the chi transgene in all generations tested so far (including fourth generation). There was no
significant difference in the relative increase in flavonol levels over
control in subsequent generations, or between hemizygous and
homozygous populations (data not shown). Fruit flavonol levels
remained elevated under open field trial and for the best lines
approached those found in yellow onions, a crop that is naturally
high in flavonols13 (Fig. 6).
The vegetative phenotype of pBBC50 plants was indistinguishable from the parental variety. Fruit peel of pBBC50-transformed
plants did not accumulate significant amounts of yellow-colored
naringenin chalcone which may explain our observation of a slight
dullness in the tone of red color in ripe fruit. Biochemical analysis
indicated that the lycopene concentration of fruit had not been
altered (data not shown).
Properties of paste produced from high-flavonol tomato fruit.
We have tested the effect of processing on the flavonol content of
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pastes manufactured from pBBC50-transformed fruit using a
benchtop scale processor, designed to mimic the factory scale operation. During the processing, peel and seeds are removed only after
the initial heating step; as a consequence, the flavonols present in
peel "leach" into the resulting paste. Concentrated paste manufactured from pBBC50-transformed fruit contained up to 1.9 mg
flavonols per gram dry weight paste, a 21-fold increase over paste
manufactured using the azygous control (data not shown). The paste
retained 65% of the total flavonol content of the fresh fruit (data not
shown), the remainder being destroyed during processing or discarded with the waste peel and seeds.
We further established that these high-flavonol pastes were indistinguishable in their taste and flavor from azygous control pastes
(data not shown).
Conclusions. Constitutive overexpression of a Petunia gene
encoding CHI in tomato resulted in elevated flavonol end products
in the fruit peel. The tomato lines contained significantly increased
levels of quercetin glycosides, and smaller but still substantial
increases in kaempferol glycosides in fruit peel. These new varieties
may offer opportunities for tomato-based products with an expanded range of potential health benefit properties.
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Figure 6. Flavonol levels of field trial transgenic fruit. Homozygous
transgenic plants were grown in controlled open field trial conditions
(1999 season, Stockton, California). Flavonols (quercetin plus
kaempferol) were extracted under hydrolyzing conditions and analyzed by
HPLC. Each bar represents 10 individual fruit (central columella and
parenchyma included, seeds removed) readings from a transgenic plant
line. Error bars representing the standard deviation of fruit in each line are
shown. Black bars (–) represent fruit of azygous control plants. Unshaded
bars (+) represent pBBC50-transformed plants.
B
diameter) from mature plants, separated fruit tissues, and whole fruit were
harvested, frozen in liquid nitrogen, and stored at –80°C until use.
Extraction and analysis of flavonoids. Flavonoids were determined either as
aglycons or as flavonoid glycosides by preparing acid-hydrolyzed and nonhydrolyzed extracts, respectively. Frozen tissues were ground to a fine powder
and lyophilized before extraction. Preparation of acid-hydrolyzed extracts was
performed according to Hertog et al.17 with some modifications. Lyophilized
material (50 mg) was hydrolyzed in 1.6 ml of 62.5% methanol and 0.4 ml 6 M
HCl for 60 min at 90°C. Extracts were cooled on ice, diluted with 2 ml of 100%
methanol, and sonicated at room temperature for 5 min. For nonhydrolyzed
extracts, 50 mg of lyophilized material was extracted into 4 ml of 70%
methanol by sonication at room temperature for 30 min. Extracts were filtered
through 0.2 µm poly-tetrafluoroethylene (PTFE) filters before HPLC.
HPLC was carried out on an HP1100 system (Hewlett Packard,
Waldbronn, Germany) using a Nova-pak C18 (3.9 × 150 mm, particle size
4 µm) reverse-phase analytical column (Waters chromatography, Milford,
MA). A photodiode array detector (Hewlett Packard) was used to record
online spectra (from 200 nm to 550 nm) of compounds eluting from the column. HPLC separation of acid-hydrolyzed extracts was carried out under
isocratic conditions of 25% acetonitrile in 0.1% trifluoroacetic acid (TFA) at
a flow rate of 0.9 ml/min. Nonhydrolyzed extracts were separated using a gradient of acetonitrile in 0.1% TFA, at a flow rate of 1.0 ml/min: 5–25% linear
in 30 min, then 25–30% in 5 min and 30–50% in 2 min, followed by a 3 min
washing with 50% acetonitrile in 0.1% TFA. Peak purity, identification, and
integration were carried out on Hewlett Packard Chemstations software version A.04.02 using commercially available flavonoid standards (Apin
Chemicals, Ltd., Abingdon, Oxon, UK). The detection limit using this system
was 0.05 µg flavonoid/ml extract, corresponding to ∼5 mg/kg dry weight and
0.5 mg/kg fresh weight tomato.
RNA extraction and analysis. RNA was isolated according to the protocol of
van Tunen et al16. For RNA gel blot analysis 10 µg of total RNA was loaded on
formaldehyde agarose gels18 and electrophoresed overnight at 25 V. Separated
RNA was blotted onto Hybond N+ membrane (Amersham Pharmacia
Biotech, Bucks, UK). Petunia hybrida cDNA fragments encoding the
flavonoid biosynthetic genes chs-a, chi, and fls were radiolabeled with 32P and
purified using a RadPrime Labelling system (Gibco Life Technologies, Paisley,
UK). Blots were hybridized overnight at 55°C and washed three times in
2 × sodium saline citrate, 1% sodium dodecyl sulfate, 55°C, 30 min before
being subjected to autoradiography. Equal loading was checked by UV imaging of ribosomal RNA bands in all sample lanes before blotting.
Figure 5. Chromatogram recorded at 370 nm, of nonhydrolyzed peel of
control and pBBC50-transformed fruit. Tomatoes transformed with
(A) control plasmid pSJ89 or (B) pBBC50 (plant no. C87). The major
peaks corresponding to rutin (R), isoquercitrin (IQ), kaempferol
rutinoside/quercetin glycoside (KR/QG)(co-eluting compounds), and a
putative kaempferol glycoside (KG) are marked.
Experimental protocol
Plasmid construction. The Petunia chi gene was amplified from plasmid
pMIP41, which contains the complete chi complementary DNA (cDNA)
from P. hybrida inbred line V30 (ref.14). The chi coding sequence was cloned
as a BamHI/SalI fragment into pFLAP10, a pUC derivative, containing a
fusion of the double CaMV 35S promoter (Pd35S) and the Agrobacterium
tumefaciens nos terminator (Tnos). A synthetic adapter fragment, consisting
of a multiple cloning site (PacI/EcoRI/HindIII/AscI), was ligated into plasmid
pGPTV-KAN (ref. 15) digested with EcoRI/HindIII, replacing the gusA-Tnos
gene. The resulting plasmid was designated pBBC3. The Pd35S-chi-Tnos construct was transferred as a PacI/AscI fragment into pBBC3 and the resulting
binary plasmid designated pBBC50. A GPTV-based binary plasmid (pSJ89)
containing the β-glucuronidase gene under control of the CaMV 35S promoter and the nos terminator signal was used as a control plasmid.
Generation of transgenic plants. Plasmids pBBC50 and pSJ89 were transferred
to A. tumefaciens strain LBA4404, which was then used to transform tomato
(var. FM6203, Unilever commercial variety) leaf disks according to standard
protocols16. The transgenic status of kanamycin-positive explants was confirmed by PCR and Southern blot analysis. Plants were transferred to soil and
grown in a glasshouse with a 16 h photoperiod and a 23°C day/18°C night temperature. Plants were allowed to self-pollinate for the production of subsequent
generations. Homozygous lines were selected on the basis of PCR screening.
Harvest of transgenic material. Fully red ripe glasshouse-grown fruits
were harvested between 15 and 21 days post-breaker stage (breaker stage
corresponds to the first flush of color on the green fruit). The outer layer of
2 mm thick (i.e. cuticula, epidermal layer plus some subepidermal tissue)
was separated from the fruit and classified as peel. The columella, jellylike
parenchyma, and seeds were removed and the remainder of the fruit was
classified as flesh. Field trial fruits were harvested when visually ripe (i.e.,
fully red). The columella, parenchyma, and seeds were removed. The
remainder of the fruit was classified as whole fruit. Young leaves (∼3–5 cm
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Acknowledgments
The authors wish to thank Dr. Adel Elsheikh and Dr. Jinguo Hu (Lipton
Innovation Centre, Stockton, CA) for their assistance with transgenic field trials. We also thank Bob Cowper, Carl Jarman, Tracey Macdonald, and
Katherine Redwood (URC, Sharnbrook, UK) for their technical contributions.
Received 16 October 2000; accepted 23 February 2001
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