Download A Fruit-Specific Putative Dihydroflavonol 4

Plant Physiol. (1998) 117: 711–716
Rapid Communication
A Fruit-Specific Putative Dihydroflavonol 4-Reductase
Gene Is Differentially Expressed in Strawberry during the
Ripening Process1
Enriqueta Moyano, Ignacio Portero-Robles, Nieves Medina-Escobar, Victoriano Valpuesta, Juan Muñoz-Blanco,
and José Luis Caballero*
Departamento de Bioquı́mica y Biologı́a Molecular e Instituto Andaluz de Biotecnologı́a, Facultad de Ciencias,
Universidad de Córdoba, Avda. San Alberto Magno s/n, 14071-Córdoba, Spain (E.M., I.P.-R., N.M.-E.,
J.M.-B., J.L.C.); and Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Málaga,
29071 Málaga, Spain (V.V.)
A cDNA clone encoding a putative dihydroflavonol 4-reductase
gene has been isolated from a strawberry (Fragaria 3 ananassa cv
Chandler) DNA subtractive library. Northern analysis showed that
the corresponding gene is predominantly expressed in fruit, where
it is first detected during elongation (green stages) and then declines
and sharply increases when the initial fruit ripening events occur, at
the time of initiation of anthocyanin accumulation. The transcript
can be induced in unripe green fruit by removing the achenes, and
this induction can be partially inhibited by treatment of de-achened
fruit with naphthylacetic acid, indicating that the expression of this
gene is under hormonal control. We propose that the putative
dihydroflavonol 4-reductase gene in strawberry plays a main role in
the biosynthesis of anthocyanin during color development at the
late stages of fruit ripening; during the first stages the expression of
this gene could be related to the accumulation of condensed tannins.
Flavonoids are a large family of secondary metabolites
widespread among plants and involved in many processes.
Anthocyanins, one class of flavonoid pigments, are the
main pigments in flowers and fruits, where they serve as
insect and animal attractants. Anthocyanin biosynthesis
has been extensively investigated in several plant species.
The structural and regulatory genes of this metabolic pathway have been cloned and their spatial and temporal expression studied (Koes et al., 1994; Holton and Cornish,
1995).
Strawberry (Fragaria spp.) is a commercially important,
nonclimateric soft fruit in which color is a crucial quality
factor. Its pigments have been partly characterized and
include anthocyanins, pelargonidin, cyanidin, and proanthocyanins (Macheix et al., 1990). However, the
anthocyanin-synthesis pathway in strawberry is poorly understood, particularly at the molecular level. Anthocyanins
1
This work was supported by Comisión de Investigación Cientifica y Técnica, Spain (no. ALI97-0836-CO3-03), Junta de Andalucı́a, Spain (no. CVI 0115), and Agriculture and Fisheries Programme, European Union (grant no. FAIR-CT97-3005).
* Corresponding author; e-mail [email protected]; fax 34 –
957–218606.
accumulate during the ripening process, which is accompanied by a decrease in firmness and chlorophyll content
(Perkin-Veazie, 1995). During this process there is an induction of the activities of PAL, the first enzyme of the
general phenylpropanoid pathway, and UDP-Glc:flavonoid O3-glucosyltransferase, the last enzyme of this
pathway.
DFR catalyzes the last common step in the flavonoidbiosynthesis pathway leading to anthocyanins and proanthocyanidins (condensed tannins). In an NADPHdependent reaction the enzyme reduces dihydroflavonols
to 3,4-cis-leucoanthocyanidins, the immediate precursors of
the anthocyanidins. DFR genes have been isolated from
several higher plants (Beld et al., 1989; Kristiansen and
Rohde, 1991; Helariutta et al., 1993; Bongue-Bartelsman et
al., 1994; Sparvoli et al., 1994; Rosati et al., 1997). The
crucial role of this gene in anthocyanin biosynthesis has
been demonstrated by transformation of petunia with a
heterologous DFR gene, which led to novel or increased
flower pigmentation (Meyer et al., 1987; Helariutta et al.,
1993; Tanaka et al., 1995).
In this paper we report the cloning and characterization
of a strawberry (Fragaria 3 ananassa cv Chandler) cDNA
gene (njjs 24) encoding an amino acid sequence that shows
a high homology to DFR from higher plants. This is the first
time, to our knowledge, that a predominantly fruitexpressed, putative DFR gene has been cloned from a soft
fruit such as strawberry. Studies of the expression pattern
of this gene have been carried out. The information provided here could be useful for genetic transformation programs aimed at changing fruit color by modifying anthocyanin synthesis.
MATERIALS AND METHODS
Strawberry (Fragaria 3 ananassa cv Chandler) fruit was
harvested at different developmental stages: small-sized
green fruits (G1), middle-sized green fruits (G2), full-sized
Abbreviations: DFR, dihydroflavonol 4-reductase; PAL, Phe
ammonia-lyase.
711
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712
Moyano et al.
green fruits (G3), white fruits with green achenes (W1),
white fruits with red achenes (W2), turning-stage fruits (T),
and full-ripe red fruits (R).
DFR cDNA Cloning
The putative DFR cDNA from strawberry was isolated
from a cDNA subtractive library by the magnet-assisted
subtraction technique-PCR-Southern blot differential
screening method previously described (Medina-Escobar
et al., 1997b).
RNA Isolation and Northern Analysis
Total RNA from fruits at the stages indicated and from
roots, leaves, flowers, and stolons was isolated according to
the method of Manning (1991). Northern analysis was as
described by Medina-Escobar et al. (1997a, 1997b). A cDNA
corresponding to 18S rRNA was used as a control for equal
loading of the RNA samples. The probes were radiolabeled
to a specific activity of approximately 108 cpm mg21 using
a commercial random-priming kit (Pharmacia).
Auxin Treatment
Achenes from two sets of G2-stage strawberry fruits
were carefully removed in the growing plant using the tip
of a scalpel blade. One set of de-achened fruits was treated
with the synthetic auxin NAA in a lanolin paste at 1 mm
NAA in 1% (v/v) DMSO. The other set (the control group)
was treated with the same paste without NAA. Both treatments were applied over the whole fruit surface. Fruits
were harvested 0, 24, 48, 72, and 96 h after treatment and
immediately frozen in liquid N2 and stored at 280°C.
DNA Isolation and Southern Analysis
Strawberry genomic DNA was extracted from achenes
removed from W1-stage strawberry fruits as described by
Medina-Escobar et al. (1997a). The DNA (4 mg) was digested with the restriction enzymes BamHI, BglII, EcoRI,
and HindIII. Southern analysis was performed according to
the method of Medina-Escobar et al. (1997a).
DNA Sequencing and Analysis of Sequences
DNA was sequenced by the dideoxy chain-termination
method using T3, T7, and specific primers within the
cDNA insert. An automated DNA sequencer (Prism,
Perkin-Elmer) and a sequencing kit (Perkin-Elmer) were
used to generate the sequence data. Sequences were analyzed using the TFASTA, FASTA, BESTFIT, COMPARE,
and DOTPLOT programs from the Genetics Computer
Group (Madison, WI) package (version 9.0; December 1996;
Devereux et al., 1984) and compared with the GenBank
(release 100, April 1997), EMBL (release 50.0, March 1997),
PIR (release 52.0, March 1997), and SWISPROT (release
34.0, November 1996) databases.
Plant Physiol. Vol. 117, 1998
RESULTS
Sequence Analysis of the njjs24 cDNA Clone and the
Encoded Protein
The cDNA corresponding to clone njjs24 was sequenced
to completion. The nucleotide and the predicted amino
acid sequences are shown in Figure 1. The cDNA insert is
1320 bp long and presents an open reading frame that
encodes a protein of 341 amino acid residues, with a predicted molecular mass of 38.9 kD and a pI of 6.78. Two
possible initiation codons are identified in the njjs24 cDNA
sequence (nucleotide positions 15 and 87; Fig. 1). DOT
PLOT comparison (data not shown) between the njjs24
cDNA and that of higher plant DFRs allowed us to propose
the first ATG as the initiation codon for the strawberry
njjs24 predicted protein. Moreover, this initiation codon is
more closely related to the consensus sequence of initiation
codons of higher plants, ATGGCT (Gallie, 1993).
The clone has a 39-untranslated region of 283 bp, including the stop codon and 31 residues of the poly(A1) tail. A
consensus plant polyadenylation sequence, AATAAA, is
also found in this region (nucleotide position 1255) to an
upstream distance (26 nucleotides) of the cleavage and
polyadenylation site in the range of the consensus distance
(10–30 nucleotides) for eukaryotic genes. In addition, 59
upstream of this polyadenylation site there is a TAG stop
codon that is preferentially utilized in higher plants (Gallie,
1993).
Sequence analysis and comparison with sequences in the
databank revealed high identity of this strawberry nucleotide sequence (data not shown) and the deduced amino
acid sequence of higher-plant DFR genes previously cloned
(Fig. 2). The higher values for identity and similarity were
to DFR proteins from Rosa hybrida (83.6 and 87.6%, respectively), Vitis vinifera (74.2 and 81.4%, respectively), Arabidopsis thaliana (72.3 and 78.1%, respectively), Dianthus
caryophyllus (70.6 and 77.7%, respectively), and Antirrhinum
majus (68.9 and 77.6%, respectively). In addition, the strawberry njjs24 deduced protein showed significant homology
to other DFRs from Gerbera hybrida, Medicago sativa, Petunia
hybrida, Lycopersicon esculentum, Gentiana triflora, Forsythia 3 intermedia, Callistephus chinensis, Hordeum vulgare,
Oryza sativa, Ipomea purpurea, and Zea mays (with identities
and similarities ranging from 64.0 to 69.0% and 76.0 to
78.0%, respectively).
An N-terminal NADP-binding domain, previously proposed as the dinucleotide-binding fold of NADP(H) and
NAD(H)-dependent reductases and dehydrogenases (Lacombe et al., 1997), is also present in the strawberry putative DFR protein (Fig. 1).
Tissue and Developmental Gene Expression
The spatial and temporal expression pattern of this
strawberry putative DFR gene has been studied. Total RNA
isolated from fruits (receptacle plus achenes) at different
development stages and from roots, leaves, flowers, and
stolons were analyzed by northern hybridization (Fig. 3). A
transcript of about 1.3 kb was specifically detected in fruits,
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Strawberry Dihydroflavonol 4-Reductase Gene and Fruit Ripening
713
Because achenes are the true fruits embedded in the
epidermal layer of the receptacle in strawberry (PerkinVeazie, 1995), expression of the njjs24 gene was separately
followed in the achenes and the receptacle (de-achened
fruit; Fig. 4). Transcripts were present in the receptacle but
were not detectable in achenes (data not shown).
Studies of the effect of auxin in njjs24 gene expression
were performed in de-achened green strawberry fruits supplied with external NAA in a lanolin paste. A clear increase
in the expression of the njjs24 gene was found in deachened G2 fruits. However, a reduction in the expression
of this gene was observed in de-achened G2 fruits treated
with NAA, and practically no expression was found in
normal G2 fruits (with achenes; Fig. 5). The inability to
completely revert njjs24 gene expression by NAA in G2
de-achened fruit has also been observed in other ripeningrelated genes in strawberry (Medina-Escobar et al., 1997a)
Figure 1. Nucleotide and deduced amino acid sequence from strawberry cDNA clone njjs24 (accession no. AF029685). Initiation and
stop codons are shown in bold. The putative polyadenylation sequence is underlined. Asterisks (*) indicate amino acid residues
conserved in the hydroxysteroid dehydrogenase/DFR superfamily
(Baker and Blasco, 1992). The box located in the N-terminal region
shows the putative NADP-binding domain. A second box shows the
stretch thought to define the substrate specificity of the DFR enzyme
(Beld et al., 1989).
showing two peaks during the developmental stages. Transcripts slightly increased in fruits during the elongation/
green stages and then declined in the W1 stage. A second
increase in the transcript level was observed in the W2
stage when the initial ripening events began. The high
expression level continued throughout the T and R stages.
Accumulation of anthocyanins occurs parallel to ripening in strawberry fruits (Perkin-Veazie, 1995). We evaluated the anthocyanin content of ripening fruits of cv Chandler, which were undetectable during the green and W1
stages but rapidly accumulated in the T and R stages
(results not shown), reaching a value of approximately 32
mmol fruit21 at the R stage. This increase correlated well
with the second increase of the njjs24 gene expression
observed from the W2 to the R stage.
Figure 2. Comparison of the amino acid sequences of the putative
strawberry DFR with that of higher plants. Identical and conservative
amino acids found in all DFR sequences are shown in black; those in
four or more sequences are shown in gray. The amino acid region
determining the substrate specificity is underlined. The accession
numbers are X15536 for A. majus (ampallid); X15537 for P. hybrida
(phybrida); AF029685 for F. ananassa (dhfrt7); D85102 for R. hybrida
(rosa); X75964 for V. vinifera (vvinifera); P51102 for A. thaliana
(athaliana); and Z67983 for D. caryophyllus (dcaryophyl). Alignment
was performed using the GenDoc program (version 2.0.004, 1997)
by K.B. Nicholas and H.B. Nicholas, Jr.
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714
Moyano et al.
Figure 3. Northern analysis of 20 mg of total RNA isolated from
pooled samples of G1, G2, G3, W1, W2, T, and R strawberry fruit
stages and from roots (Rt), leaves (L), flowers (F), and stolons (St). The
size of hybridizing transcript is indicated in kilobase pairs. A, Hybridization was to a 32P-labeled njjs24 probe. B, The same blot was
hybridized with a 32P-labeled 18S RNA probe used as a control.
and was explained by an incomplete penetration and/or
degradation in the receptacle of the NAA.
Southern-Blot Analysis
Strawberry genomic DNA was digested with the restriction enzymes BamHI, BglII, EcoRI, and HindIII and analyzed by Southern-blot hybridization using the same probe
previously utilized in northern-blot studies (Fig. 6). Several
hybridization fragments above 5.4 kb were detected in the
BamHI, BglII, and EcoRI digestions. No restriction sites
were found in the strawberry njjs24 cDNA sequence for
Figure 4. Northern analysis of 20 mg of total RNA isolated from
receptacle (de-achened fruit) at the same developmental stages indicated in Figure 3. A and B are as described in Figure 3.
Plant Physiol. Vol. 117, 1998
Figure 5. Effects of removing achenes and treatment with auxin on
putative strawberry DFR gene expression. Northern analysis of total
RNA (20 mg) isolated from G2-stage strawberry fruit at 0 h (lane 1),
24 h (lane 2), 48 h (lane 3), 72 h (lane 4), and 96 h (lane 5) after
removing the achenes (Ac). Lane 6, G2-stage de-achened fruit treated
with NAA at 96 h. Lane 7, G2-stage strawberry fruit with achenes. A
and B are as described in Figure 3.
these enzymes. Furthermore, only a restriction site was
found in the njjs24 cDNA sequence for HindIII; therefore,
at least two hybridization fragments were expected. However, six hybridization fragments of sizes ranging between
1.5 and 7.6 kb were detected in the HindIII digestion. The
long fragments observed can be due either to a small
multigenic family or to the ploidy level of the strawberry
cultivar (octoploid).
DISCUSSION
Although there is no functional proof of the role of the
njjs24 gene product, we propose that this gene encodes a
DFR enzyme. The high level of homology to the DFR
sequences from the databank, combined with fruit-specific
and ripening-related expression, supports this assignment.
Figure 6. Southern blot of strawberry genomic DNA digested with
several restriction enzymes. Total DNA (4 mg) was extracted from
achenes, electrophoresed, blotted, and hybridized with the 32Plabeled njjs24 probe.
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Strawberry Dihydroflavonol 4-Reductase Gene and Fruit Ripening
Furthermore, the presence of an N-terminal NADP-binding
domain, common to other reductases, adds further support. The njjs24 gene showed the highest sequence homology at both the nucleotide and the amino acid level to that
of R. hybrida, which is understandable since strawberry
belongs to the Rosaceae family. Next in homology level
was the DFR from V. vinifera, the fruit of which, like that of
strawberry, is considered nonclimacteric.
DFR is encoded either by a small multigene family (Beld
et al., 1989; Helariutta et al., 1993) or by only one gene
(Kristiansen and Rohde, 1991; Bongue-Bartelsman et al.,
1994; Sparvoli et al., 1994; Rosati et al., 1997). In strawberry
the hybridization pattern suggests that DFR is probably
encoded by a small family of genes. The clear differences in
band intensity among the DFR-hybridizing fragments suggests the presence of other genes with different homology
to njjs24, but the confidence level for statements concerning
gene number is reduced when the source of the DNA is an
octoploid (such as Fragaria 3 ananassa cv Chandler). Also,
the two peaks in expression observed during fruit development and ripening could be explained by the existence
of two differentially regulated DFR genes. This issue remains to be further investigated.
The enzymatic activities of PAL and UDP-Glc:flavonoid
O3-glucosyltransferase in the biosynthetic pathway leading
to pelargonidin-3-glucoside (the major anthocyanin pigment of ripe strawberry) increase during strawberry fruit
ripening (Perkin-Veazie, 1995). Parallels between PAL activity and anthocyanin content have specifically been reported in the strawberry cv Tillikum (Cheng and Breen,
1991). It is noteworthy that two peaks of PAL activity have
been reported in strawberry fruits, one in green fruits and
one in nearly ripe fruits (Cheng and Breen, 1991). Whereas
the second activity peak has been associated with the anthocyanin accumulation that occurs in ripe fruits, the first
peak has been explained by its involvement in the synthesis of other flavonoids (condensed tannins) and phenolics
that takes place during early fruit development (Macheix et
al., 1990; Cheng and Breen, 1991).
The two peaks of DFR transcripts reported here in strawberry fruits during growth and ripening seem to be coincident to the PAL activity peaks previously reported in this
fruit. Therefore, we propose that the first DFR expression
increase is probably associated with the synthesis of condensed tannins in green fruits, and the ripening-associated
increase functions in the biosynthesis of anthocyanins.
Close association between DFR expression and condensed
tannin accumulation has been reported in young V. viniferis
berries (Boss et al., 1996) and in root cultures of bird’s foot
trefoil (Bavage et al., 1997). There are also abundant reports
in the literature showing that DFR gene expression is spatially and developmentally correlated with the anthocyanin
accumulation pattern in different plant tissues (Beld et al.,
1989; Jackson et al., 1992; Huits et al., 1994; Tanaka et al.,
1995; Rosati et al., 1997).
It has been proposed that auxin stimulates expansion of
the receptacle and inhibits ripening in strawberry fruit
(Manning, 1993; Perkin-Veazie, 1995). Moreover, an induction pattern of mRNA population in immature de-achened
fruit was found to be reverted by the application of the
715
synthetic auxin NAA to the de-achened fruit receptacle,
supporting a relationship between ripening-induced gene
expression and the absence of auxin (Perking-Veazie,
1995). Some unidentified fruit-specific strawberry genes
have been shown to be under the control of auxins (Reddy
and Poovaiah, 1990; Reddy et al., 1990). More recently, it
has been reported that a fruit-specific strawberry pectate
lyase gene, the expression of which was only induced in
the W2 to R stages, was negatively regulated by auxins
(Medina-Escobar et al., 1997a). Similarly, the expression of
the fruit-specific strawberry DFR was clearly induced in
de-achened G2 fruit, and this induction was partially reverted by NAA application to de-achened receptacles.
These results indicate that DFR expression in strawberry is
either directly or indirectly under the control of internal
auxin, as has been proposed for other ripening-related
strawberry genes (Reddy and Poovaiah, 1990; Reddy et al.,
1990; Medina-Escobar et al., 1997a).
In summary, we have isolated and characterized a strawberry cDNA clone, njjs24, which corresponds to an mRNA
probably encoding a DFR protein. Experiments with antisense RNA are in progress to elucidate the involvement of
this strawberry DFR gene in phenylpropanoid metabolism
during fruit development and ripening.
ACKNOWLEDGMENTS
The use of the equipment at the Instituto Andaluz de Biotecnologı́a, Andalucı́a, Spain, is gratefully acknowledged. E.M. and
N.M.-E. thank the Ministerio de Educación y Ciencia, Spain, for a
postdoctoral research contract and predoctoral fellowship, respectively. The authors also thank J.M. López-Aranda and J.J. Medina
(Centro de Investigaciones de Desarrollo Agrario “Churriana”
Málaga, Spain) for their help with strawberry field cultivation.
Received November 11, 1997; accepted February 25, 1998.
Copyright Clearance Center: 0032–0889/98/117/0711/06.
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