Premature and ectopic anthocyanin formation by silencing of

anthocyanidin reductase in strawberry (Fragaria 9ananassa)

Thilo C. Fischer
1
, Beate Mirbeth
1
, Judith Rentsch
2
, Corina Sutter
3
, Ludwig Ring
3
, Henryk Flachowsky
2
,
Ruth Habegger
3
, Thomas Hoffmann
3
, Magda-Viola Hanke
2
and Wilfried Schwab
3
1
Plant Biochemistry and Physiology, Ludwig-Maximilians-University Munich, Grohadernerstr 24, D-82152, Planegg-Martinsried, Germany;
2
Julius Kuhn-Institute Federal Research
Centre for Cultivated Plants, Institute for Breeding Research on Horticultural and Fruit Crops, Pillnitzer Platz 3a, 01326 Pillnitz, Dresden, Germany;
3
Biotechnology of Natural Products,
Technical University Munich (TUM), Liesel-Beckmann-Str 1, D-85354, Freising, Germany
Authors for correspondence:
Thilo C. Fischer
Tel: +49 89 2180 74754
Email: thilo.scher@biologie.
uni-muenchen.de
Wilfried Schwab
Tel: +49 8161 71 74754
Email: schwab@wzw.tum.de
Received: 10 June 2013
Accepted: 19 August 2013
New Phytologist (2013)
doi: 10.1111/nph.12528
Key words: anthocyanidin reductase (ANR),
anthocyanin, epicatechin, avonols,
proanthocyanidins, RNAi, strawberry
(Fragaria 9ananassa).
Summary

Strawberry (Fragaria 9ananassa) is a fruit crop with a distinct biphasic avonoid biosynthe-
sis. Whereas, in the immature receptacle, high levels of proanthocyanidins accumulate, which
are associated with herbivore deterrence and pathogen defense, the prominent color-giving
anthocyanins are primarily produced in ripe fruits helping to attract herbivores for seed
dispersal.

Here, constitutive experimental down-regulation of one branch of proanthocyanidin

biosynthesis was performed.

As a result, the proportion of epicatechin monomeric units within the proanthocyanidin

polymer chains was reduced, but this was not the case for the epicatechin starter unit. Short-
ened chain lengths of proanthocyanidins were also observed. All enzymatic activities for the
production of color-giving anthocyanins were already present in unripe fruits at levels allow-
ing a striking red anthocyanin phenotype in unripe fruits of the RNAi silencing lines. An imme-
diately recognizable phenotype was also observed for the stigmata of owers, which is
another epicatechin-forming tissue.

Thus, the down-regulation of anthocyanidin reductase (ANR) induced a redirection of the

proanthocyanidin pathway, leading to premature and ectopic anthocyanin biosynthesis via
enzymatic glycosylation as the alternative pathway. This redirection is also seen in avonol
biosynthesis, which is paralleled by higher pollen viability in silencing lines. ANRi transgenic
lines of strawberry provide a versatile tool for the study of the biological functions of proanth-
ocyanidins.
Introduction
In strawberry (Fragaria 9ananassa), avonoids are secondary
metabolites that are of interest because of their multiple biologi-
cal functions in plants as well as in human consumption. In ripe
fruits, the bright red anthocyanin pelargonidin and, to a lesser
degree, the dark red cyanidin (dependent on cultivar) are decisive
for fruit attractiveness. Flavonols are also present and their
health-promoting effects have been broadly discussed (Graf et al.,
2005; Munoz et al., 2011). Monomeric and polymeric avan-
3-ols mainly occur in unripe fruits (reviewed in Flachowsky et al.,
2011). The major avonoid genes of strawberry have been
cloned, characterized with respect to general substrate specicity
and the gene copy numbers of the octoploid strawberry have been
studied (Almeida et al., 2007) (Fig. 1). For some genes and their
respective enzymes, more specic functions have been studied,
such as for avonol-O-glucosyltransferases (Griesser et al., 2008a)
and dihydroavonol-4-reductase/avanone-4-reductase (Fischer
et al., 2003). In addition, the genome of the diploid woodland
strawberry Fragaria vesca has been elucidated (Shulaev et al.,
2011), providing a broad base of information on orthologous
and paralogous genes from one of the precursors of cultivated
octoploid strawberry.
Flavan-3-ols are a diverse class of avonoids. Monomeric
avan-3-ols (catechins in general) differ in stereochemistry at the
C3 hydroxyl group in the C-ring. 2,3-trans-Catechin (catechin in
the strict sense) is formed via the reduction of leucocyanidin by
leucoanthocyanidin-4-reductase (LAR) (Tanner & Kristiansen,
1993; Tanner et al., 2003), whereas 2,3-cis-catechin (epicatechin)
occurs by the reduction of cyanidin by anthocyanidin reductase
(ANR) (Xie et al., 2003) (Fig. 1). Flavan-3-ols of both stereo-
chemistries are found with different numbers of B-ring hydroxy-
lations, depending on the plant source. Most common are the
3,4-dihydroxylated avan-3-ols catechin and epicatechin,
whereas the 4-monohydroxylated equivalents are afzelechin and
epiafzelechin, which are also formed in strawberry. By contrast,
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the 3,4,5-trihydroxylated gallocatechin and epigallocatechin are
not known from strawberry. Proanthocyanidins are polymeric
avan-3-ols and are supposed to be formed from such mono-
meric building blocks. Starter and extension units are connected
by an as yet unknown mechanism. Almost no free epicatechin is
found in strawberry, but it is quite common as a subunit in the
proanthocyanidins (Buenda et al., 2010). Proanthocyanidins
show much structural variation with respect to the kinds of build-
ing blocks (C3-hydroxy group stereochemistry and B-ring
hydroxylation), chain length and stereochemistry of the interav-
anoid bond. Specic biological effects may be expected, but their
analysis is hampered by demanding analytics and difcult
preparative isolation.
Experimental manipulation of avan-3-ol metabolism has
been performed with a number of systems. Mutation, silencing
and overexpression of transcription factors (tt2, tt8, ttg1) have
been performed to identify specic functions with the system
Arabidopsis thaliana (Debeaujon et al., 2003). For the Rosaceae
family member apple (Malus 9domestica), which is related to
strawberry, Myb-type transcription factors for (pro)anthocyanin
biosynthesis have been identied (Takos et al., 2006; Ban et al.,
2007; Espley et al., 2007), whereas, in strawberry, the factor
FaMYB1 has been shown to control anthocyanin biosynthesis
(Aharoni et al., 2001) and to suppress anthocyanin and to
enhance avan-3-ol formation (Salvatierra et al., 2013). In gen-
eral, R2R3 MYB transcription factors are involved in the regula-
tion of (pro)anthocyanin biosynthesis in Rosaceae (Lin-Wang
et al., 2010). Overexpression of the Leaf color (Lc) gene, a heterol-
ogous Myc-type transcription factor from maize, in apple led to
enhanced levels of specic proanthocyanidins of up to 100-fold
(Li et al., 2007; Flachowsky et al., 2009). The last step before the
nal branching point between epicatechin and anthocyanin bio-
synthesis is catalyzed by anthocyanidin synthase (ANS), which is
responsible for anthocyanidin formation. Subsequently, avo-
noid-3O-glycosyltransferases (FGTs) or ANR performs the com-
mitted steps towards either stable anthocyanin or epicatechin and
its polymers. ANR has been overexpressed in Arabidopsis and
heterologous systems (Nicotiana) to demonstrate its function
(Xie et al., 2003). The structural genes have also been targets for
silencing approaches. In apple, the ANS gene ans has been
silenced, resulting in the expected abolishment of anthocyanin
formation, but this also led to an unexpected rise in free epicate-
chin (Szankowski et al., 2009). In strawberry, redirection of the
anthocyanin pathway to avan-3-ols was observed by the down-
regulation of an anthocyanidin glucosyltransferase in ripening
strawberry fruit (Griesser et al., 2008b).
In this work, experimental manipulation in strawberry was
performed by the silencing of ANR. In strawberry, this is at the
developmental switch between early proanthocyanidins, which
might be involved in pathogen defense (Jersch et al., 1989), and
late color-determining anthocyanins. This approach resulted in
strong unbalancing of avonoid classes and a striking anthocya-
nin phenotype in unripe fruits. Ectopic anthocyanin formation
in other epicatechin-forming organs (Hoffmann et al., 2012),
such as the stigmata of owers and root tips, was also observed.
Materials and Methods
Plant material
The octoploid strawberry (Fragaria 9ananassa Duch.) cv Senga
Sengana was used for this study. For convenience, the unripe and
ripe receptacles of strawberry are referred to as fruit in this
article, even though they are not botanically dened as such.
Preparation of silencing intron-hairpin vector construct
The pBI-ANRi construct was generated according to Hoffmann
et al. (2006). b-Glucuronidase (GUS) of pBI121 (Jefferson,
1987) was replaced by the second intron of the F. 9ananassa
enone oxidoreductase gene (AY158836, nucleotides 48864993).
A 115-bp fragment corresponding to nucleotides 32146 of
F. 9ananassa ANR mRNA (DQ664192) was inserted into the 5
and 3 arms of the intron. This ANR gene had been identied by
cDNA cloning (Almeida et al., 2007) and represents the major
expressed gene of ANR in octaploid strawberry. The resulting
plasmid was named pBI-ANRi (Supporting Information Fig. S1).
Plant transformation
Axillary shoot cultures of strawberry were used for plant trans-
formation. The plant material was propagated in vitro on shoot
proliferation medium containing Murashige and Skoog (MS)
Fig. 1 Biosynthesis of avonoids in strawberry (Fragaria 9ananassa). Only
dihydroxylated avonoids and precursors are shown; biosynthesis of
monohydroxylated avonoids occurs in parallel. ANR, anthocyanidin
reductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; C4H,
cinnamic acid-4-hydroxylase; C4L, 4-coumarate:CoA ligase; CHS,
chalcone synthase; DFR, dihydroavonol-4-reductase; FGT1, UDP-
glucose:avonoid-3-O-glucosyltransferase; FHT, avanon-3-b-
hydroxylase (syn. F3H); FLS, avonol synthase; LAR, leucoanthocyanidin
reductase; PAL, phenylalanine ammonia-lyase. Changes caused by
silencing of anthocyanidin reductase ANR are also indicated by arrows
(see the Results section).
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salts and vitamins (Murashige & Skoog, 1962) supplemented
with 0.1 mg l
1
6-benzylaminopurine (BAP), 0.01 mg l
1
indole-3-acetic acid (IAA), 30 g l
1
sucrose and 0.45% Difco
Bacto-agar (Difco, Heidelberg, Germany). Plants were grown
in a culture chamber (16 h light at 21C and 8 h dark at
16C) and subcultured every 34 wk. A single colony of the
Agrobacterium tumefaciens strain AGL0 containing the plasmid
vector pBI-ANRi was grown overnight in 2 ml of liquid Luria
Bertani (LB) medium supplemented with 100 lg l
1
rifampicin
and 100 lg l
1
kanamycin. LB plates were inoculated by
streaking 30 ll of the liquid cultures. Five plates were incu-
bated for 4 d at 28C. The bacteria were resuspended in 25 ml
of simplied induction medium (SIM) (2% sucrose, 20 mM
sodium citrate, 0.1 mM acetosyringone, 1 mM betaine hydro-
chloride, pH 5.2), and grown at 28C and 220 rpm on a hori-
zontal shaker to give a nal optical density at 600 nm (OD
600
)
of 0.81. Plant material was dipped into this bacterial suspen-
sion and was subsequently co-cultivated in the dark at 25C
on solid regeneration medium consisting of 4.4 g l
1
MS salts
and vitamins (M0222; Duchefa, RV Haarlem, the
Netherlands), 2% sucrose, 9 mM thidiazuron (TDZ), 0.9 mM
IAA and 0.3% gelrite at pH 5.7 for 3 d. Leaf explants were
washed in MSO medium containing 2.15 g l
1
MS salts
(M0221, Duchefa) and 500 mg l
1
timetin for 10 min, in dou-
ble-distilled H
2
O for 5 min and, nally, in MSO again as
described. Adventitious shoot induction was performed on
regeneration medium supplemented with 500 mg l
1
timetin
and 300 mg l
1
kanamycin. Explants were cultured in the dark
for 3 wk at 25C, and then under a 16 h : 8 h photoperiod at
the same temperature. Regenerated meristems were excised 9
12 wk after inoculation and independent transgenic clones
from a single transformation event were obtained. Transgenic
shoots were subcultured on shoot proliferation medium with
500 mg l
1
timetin and 300 mg l
1
kanamycin under a photo-
period of 16 h of light at 21C and 8 h of darkness at 16C.
Rooted plantlets were transferred into 5-cm plastic pots with
soil and acclimatized in the glasshouse. Independent T0 lines
were propagated via stolons to provide sufcient plant material
for the studies.
PCR and Southern blot analysis
Genomic DNA was extracted from in vitro leaves using a modi-
ed cetyltrimethylammonium bromide (CTAB) extraction pro-
tocol. PCRs for testing the transgenic plants for the presence of
transgenic DNA were performed in 25 ll of reaction mixture
containing 50 ng DNA, 1 9DreamTaq
TM
buffer, 0.2 mM deoxy-
nucleoside triphosphates (dNTPs), 0.5 lM of each primer
and 0.5 U DreamTaq
TM
DNA polymerase (MBI Fermentas, St.
Leon-Roth, Germany). The PCR started by initial denaturation
at 94C for 4 min, followed by 33 cycles of 30 s of denaturation
at 94C, 1 min of annealing at 56C and 1.5 min of extension at
72C, and a nal extension at 72C for 7 min. All PCRs were
performed in a MyCycler
TM
thermocycler (Bio-Rad, Hercules,
CA, USA). The PCR for detection of the nptII marker gene was
performed using the primers nptIIF (5-ACAAGATGGATTG
CACGCAGG-3) and nptIIR (5-AACTCGTCAAGAAGGC
GATAG-3), whereas, for the chimeric ANR gene, the primers
ANRF (5-CGCACAATCCCACTATCCTT-3) and ANRR
(5-TCGCCCTTAAGTCTGCTTGT-3) were used.
Southern hybridization experiments were performed using
10 lg of HindIII (MBI Fermentas)-digested DNA. The DNA
was digested with 100 U of HindIII at 37C overnight. The
restricted DNA was separated on a 0.8% agarose gel and trans-
ferred onto a nylon membrane (Roche Diagnostics, Mannheim,
Germany). PCR-amplied, digoxigenin-labeled probes from the
coding region of the nptII marker gene were generated using the
primers nptIIF/R and used for hybridization. Hybridization and
detection were performed using the ECF Random Prime Label-
ing and Detection Kit (Amersham Biosciences, Freiburg,
Germany) according to the manufacturers manual.
Histochemical analysis
Dimethylaminocinnamicaldehyde (DMACA) staining for avan-
3-ols (Feucht reaction) of tissues was performed by exposure for
1 min in 1% DMACA (w/v) in ethanol/6 M HCl (1 : 1). Ethanol
was used instead of n-butanol (W. Feucht, pers. comm.). The
specicity of the assay has been studied previously (Treutter,
1989; Hoffmann et al., 2012).
For diphenylboric acid 2-aminoethylester (DPBA) staining for
avonols, plant tissues were vacuum inltrated with a saturated
DPBA solution of 0.5% w/v DPBA (Sigma-Aldrich) and 0.1%
Triton X-100 for 30 min. Subsequently, the color was detected
under UV long-wavelength light (UV lamp, type 600352;
Waldmann, Villingen-Schwenningen, Germany). Photographs
were taken using a digital camera (D40X; Nikon, Dusseldorf,
Germany).
Gene expression analysis
Transcript levels of specic and general avonoid pathway genes
were analyzed in different tissues and developmental stages. For
ower analysis, petals, stigmata and receptacles were separated
and frozen in liquid nitrogen. Fruits were separated from green
plant material (leaves, stem) and also frozen in liquid nitrogen.
Fruits at the stages early green (G1) (achenes clearly separated
by green tissue of receptaculum) and early white (W) (starting
to lose green color and becoming white) were harvested. From
ANRi lines, fruits of the same age and size were chosen. In addi-
tion, root tips (0.5 cm) were harvested. RNA extraction was per-
formed with the RNEasy Plant MiniKit from Qiagen (Hilden,
Germany), according to the manufacturers protocol. Except for
petals, which were lyzed by buffer RLC, all samples were lyzed by
buffer RLT. Both buffers contained b-mercaptoethanol. RNA
was diluted in 30 ll (stigma, petals), 50 ll (receptacle, green
fruit) or 100 ll (white fruit) RNAse-free water. The RNA was
applied directly to quantitative PCR (qPCR) after proof of the
purity of RNA with respect to DNA (data not shown). cDNA
synthesis was performed according to recommended conditions
for Superscript reverse transcriptase (Bio-Rad). Random hex-
amer primers (New England Biolabs, Ipswich, MA, USA) and
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2050 ng of total RNA were used. qPCR was performed with a
Bio-Rad MyiQ Single Color Real-time PCR Detection System.
Reactions were set up with 10 ll cybrgreen (containing buffer,
polymerase, dNTPs and dye), 3.2 ll gene-specic primer mix
(5 lM each), 0.2 ll (all but white fruit) or 0.5 ll (white fruit)
cDNA (or water for negative control) and 6.6 ll (all but white
fruit) or 6.3 ll (white fruit) of water. Mastermixes were used to
avoid inaccuracies. Every type of reaction was performed at least
three times. The PCR program involved initially 3 min at 95C,
followed by 40 cycles (55 cycles for white fruit analyses) of 10 s at
95C, 30 s at 55C and 10 s at 72C. Primer sequences are given
in Table S1. The relative expression ratio was calculated and nor-
malized according to Pfaf (2001).
Liquid chromatography-electrospray ionization-mass
spectrometry
n
(LC-ESI-MS
n
) analysis
A Bruker Daltonics esquire 3000
plus
ion trap mass spectrometer
(Bruker Daltonics, Bremen, Germany) connected to an Agilent
1100 HPLC system (Agilent Technologies, Waldbronn,
Germany), equipped with a quaternary pump and a diode array
detector, was utilized for metabolite analysis. Components were
separated with a Phenomenex Luna C-18 column (150 mm
long 92.0 mm i.d., particle size 5 lm; Phenomenex, Aschaffen-
burg, Germany) that was held at 28C. High-performance liquid
chromatography (HPLC) was performed with the following
binary gradient system: solvent A, water with 0.1% formic acid;
solvent B, 100% methanol with 0.1% formic acid. The gradient
program was as follows: 030 min, 100% A to 50% A/50% B;
3035 min, 50% A/50% B to 100% B, hold for 15 min; 100%
B to 100% A in 5 min, then hold for 10 min. The ow rate was
0.2 ml min
1
. The ionization parameters were as follows: the
voltage of the capillary was 4000 V and the end plate was set to
500 V. The capillary exit was 121 V and the Octopole RF
amplitude was 150 Vpp (peak-to-peak voltage). The temperature
of the dry gas (N
2
) was 330C at a ow rate of 9 l min
1
. The
full scan mass spectra of the metabolites were measured at m/z
50800 until the ICC target reached either 20 000 or 200 ms.
Tandem MS was carried out using helium as the collision gas
(3.56 910
6
mbar) with a collision voltage of 1 V. Mass spectra
were acquired in negative and positive ionization modes. Auto-
tandem MS was used to break down the most abundant
[M+ H]
+
, [M H]

or [M+ HCOO]

ions of the different

compounds. Samples (fruit of different developmental stages,
roots, leaves) were individually frozen, lyophilized for 48 h and
homogenized with a mill (Retsch MM 200, Haan, Germany) to
a ne powder. An aliquot of 50 mg of lyophilized powder was
used for each of the three biological replicates. Non-natural
4-methylumbelliferyl glucoside (250 ll of a solution in metha-
nol, c = 0.2 mg ml
1
) was added as an internal standard (IS),
yielding 50 lg of IS in each sample. After the addition of 250 ll
methanol, vortexing and sonication for 10 min, the sample was
centrifuged at 16 000 g for 10 min. The supernatant was removed
and the residue was re-extracted with 500 ll of methanol. The
supernatants were combined, concentrated to dryness in a vac-
uum concentrator and redissolved in 35 ll of water. After 1 min
of vortexing, 10 min of sonication and 10 min of centrifugation
at 16 000 g, the clear supernatant was used for LC-MS analysis.
Each extract was injected twice (technical replicate). Metabolites
were identied by their retention times, mass spectra and product
ion spectra in comparison with the data determined for authen-
tic reference material. Phenylpropanoyl glucose esters were
enzymatically synthesized with FaGT2 (Fragaria 9 ananassa
glucosyltransferase 2) (Lunkenbein et al., 2006). Pelargonidin
3-O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-gluco-
side, kaempferol 3-O-glucuronide, kaempferol 3-O-glucoside,
catechin and epicatechin were obtained from Roth, Karlsruhe,
Germany. Proanthocyanidins and pelargonidin 3-O-glucoside-6-
O-malonate were isolated from strawberry and identied accord-
ing to Fossen et al. (2004). Statistical evaluation was performed
using independent Students t-test of SOFA Statistics (Paton-
Simpson & Associates Ltd, Auckland, New Zealand). The major
known phenolic metabolites were quantied in the positive
(anthocyanins) and negative (phenylpropanoids, avonoids) MS
mode by the IS method and were expressed as & equivalent of
dry weight assuming a response factor of 1. The metabolite
concentrations did not always lie within the linear range of the
detector and the calculation of their relative levels did not allow
immediate comparison with the absolute levels of phenolics pro-
vided by other studies, but the method offered the advantage to
obtain relative values in a short period of time which is sufcient
to compare relative metabolite levels. Signals of the compounds
were integrated in their [M+ H]
+
, [M H]

or [M+ HCOO]

ion traces. The non-natural 4-methylumbelliferyl glucoside was

used as IS for the relative quantication of the metabolites.
Moreover, this compound served as a control for the ionization
yield of the mass spectrometer and reproducibility of the reten-
tion times. Values are expressed in mg kg
1
-equivalent 4-methyl-
umbelliferyl glucoside (dry weight). Relative metabolite
quantication was performed using the DataAnalysis 3.1 and
QuantAnalysis 1.5 software (Bruker Daltonics), normalizing all
results to the IS.
Analysis of higher proanthocyanidins by benzylthiol
thiolysis
Thiolytic degradation and degree of polymerization (DP) calcula-
tions were performed according to Gu et al. (2003). Degradation
products were analyzed by high-performance liquid chromatogra-
phy-diode array detection-electrospray ionization-mass spectrom-
etry
n
(HPLC-DAD-ESI-MS
n
) consisting of a Bruker Daltonics
esquire 3000
plus
ion trap mass spectrometer (Bruker Daltonics)
connected to an Agilent 1100 HPLC system (Agilent Technolo-
gies), equipped with a quaternary pump and a diode array detec-
tor. Components were separated with a Phenomenex Luna C-18
column (150 mm long 92.00 mm i.d., particle size 5 lm; Phe-
nomenex) that was held at 28C. HPLC was performed with the
following binary gradient system and gradient: solvent A, water
with 0.1% formic acid; solvent B, methanol with 0.1% formic
acid; 030 min, 100% A to 50% A/50% B; 3035 min, 50% A/
50% B to 100% B, hold for 15 min; 100% B to 100% A in
5 min, then hold for 10 min. The ESI voltage of the capillary was
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Research
New
Phytologist 4
set to 4000 V and the end plate to 500 V. Nitrogen was used
as dry gas at a temperature of 330C and a ow rate of 9 l min
1
.
The capillary exit was 121 V and the Octopole RF amplitude was
150 Vpp. Tandem MS was carried out using helium as the colli-
sion gas (3.56 910
6
mbar) with a collision voltage of 1 V. Spec-
tra were acquired in positive and negative ionization mode.
Components were identied by their retention times, mass spec-
tra and product ion spectra in comparison with data determined
for authentic reference materials. Quantication was performed
by UV detection at 280 nm.
Ascorbic acid content
Ascorbic acid was quantied with the reectometer RQex

10
Reectoquant

(Merck KGaA, Darmstadt, Germany) according

to the protocol described by Pantelidis et al. (2007) with minor
modications. Fruit samples of 200 g were mixed with 200 ml of
oxalic acid (1%), homogenized for 1 min and centrifuged for
3 min at 1500 g; 2.5 ml of the supernatant were mixed with
2.5 ml of oxalic acid (1%) and supplemented with 250 mg of
polyvinylpolypyrrolidone (PVP) to remove phenols, and three
drops of H
2
SO
4
(25%) to reduce the pH 1. Results were
expressed as milligrams of ascorbic acid per 100 ml of the super-
natant supplemented with PVP and H
2
SO
4
.
Pollen viability test
The evaluation of pollen viability was performed as described by
Flachowsky et al. (2007). Anthers of glasshouse owers were col-
lected, air dried for 3 d at room temperature and stored at
80C. The number of vital and dead pollen grains was counted
after incubation in 0.5% carmineacetic acid. Dead pollen grains
stay uncolored; vital pollen grains are redorange.
Determination of fruit rmness
Firmness was recorded in three independent experiments, each
with 722 fruits per genotype (depending on the availability).
Firmness was measured using the FirmTech 2 (BioWorks,
Wamego, KS, USA) by the force test option, with all fruits
squeezed from 100 to 250 g. A value of 200 g mm
1
indicates
that 200 g of force will squeeze the fruit by 1 mm. The data
were analyzed using the software package Fruitsoft 1.5v
(BioWorks).
Results
Vector construction and plant transformation
A hairpin construct was built from a fragment of ANR (Alme-
ida et al., 2007) and an intron of a strawberry enone oxidore-
ductase gene (Raab et al., 2006), and was ligated in vector
pBI121. Such hairpin constructs give rise to the production of
small interfering RNAs (siRNAs) in plants. Three transforma-
tion experiments were performed with the strawberry (Fragaria
9ananassa) cv Senga Sengana. A total of 530 wounded leaf
discs of Senga Sengana was inoculated with the A. tumefaciens
strain AGL0 carrying the binary plasmid vector pBI-ANRi.
The transformation experiments resulted in a total of 34 puta-
tive transgenic plants representing independent transformation
events, which were selected after regeneration on regeneration
medium containing 500 mg l
1
timetin and 300 mg l
1
kana-
mycin. Seventeen of these plants survived, and the rst 14
plants which developed well were propagated to establish trans-
genic lines. These 14 lines were positively tested for the pres-
ence of the transferred gene construct using the primers
nptII_F/R for nptII and ANR_F/R for the chimeric anr gene
sequence. All transgenic plants showed PCR products of the
expected sizes (Figs S2, S3). Transgene integration was tested
by Southern hybridization (Fig. S4). All lines showed hybrid-
ization products, except for line F19, which was excluded from
further investigations as a result of the contradictory results
obtained by PCR and Southern hybridization.
Gene expression analysis
Transcript levels of avonoid genes were analyzed by reverse tran-
scription-quantitative polymerase chain reaction (RT-qPCR) in
different tissues of the ANRi lines obtained from independent
transformation events. Comparison of the gene expression data
of wild-type and ANRi transgenic lines conrmed the down-regu-
lation of the ANR gene, as indicated by small normalized expres-
sion levels in various tissues, except petals. However, mRNA
levels of MYB, C4L, CHS, DFR, ANS and FGT appeared to be
increased signicantly by a factor of up to 10 in certain tissues,
and FaMYB1, PAL, C4L, CHI, DFR, FLS and FGT levels were
reduced in some other tissues (Fig. 2). The down-regulation of
ANR is also consistent for the independent transformation events
analyzed (Fig. S5).
Anthocyanin phenotype
All independent ANRi lines showed prominent visible anthocya-
nin phenotypes (Fig. 3) with anthocyanin color in unripe fruits,
stigmata, roots and stipules of shoots and stolons. Some lines pro-
duced purple-colored petals with dark purple veins. The pheno-
type clearly separated wild-type plants from ANRi transgenic
plants. Line F17, which shows the strongest anthocyanin pheno-
type, was chosen for further in-depth analysis.
Histochemical analysis
DMACA staining for (epi)catechinsproanthocyanidins allowed
histochemical analysis to compare the anthocyanin phenotype
with tissue-specic (epi)catechinproanthocyanidin formation. A
co-localization of (epi)catechinproanthocyanidin formation in
the untransformed control (stigmata and cortex) and anthocyanin
up-regulation in ANRi lines were found (Fig. 4). This was most
clearly seen in the stigmata of the owers. DMACA staining in
the ANRi lines is probably a result of the still active catechin bio-
synthetic pathway, as LAR expression was not affected in the
ANRi lines (Fig. 1).
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Histochemical analysis for avonols in strawberry fruit was
performed by application of DPBA and imaging under UV light.
Strong and uniform up-regulation of avonols in the entire fruit
of ANRi lines in comparison with the untransformed control was
found (Fig. 5). Vascular bundles were strongly colored yellow.
Metabolic proling
Metabolite proling of the ANRi lines (F22, F21, F17, F15 and
F14) and the control was performed by LC-ESI-MS
n
analysis in
various tissues and at different fruit ripening stages. In transgenic
strawberry fruit, the anthocyanins pelargonidin and cyanidin glu-
coside were already detected in fruits of the G1 stage, unlike the
wild-type, where anthocyanins were rst found in fruits of the
white stage (Fig. 6). Cyanidin glucoside (3001600 ppm) pre-
vailed in immature fruits, whereas pelargonidin glucoside was the
predominant anthocyanin in ripe fruits of ANRi lines. It appears
that the capacity for 3-hydroxylation of the avonoid skeleton is
limited during strawberry fruit ripening, as the level of pelargoni-
din glucoside (up to 12 000 ppm) exceeded the concentration of
cyanidin glucoside by a factor of 7.5 in the red ripening stage. In
this stage, the levels of cyanidin glucoside in ANRi plants were
similar to those of control fruits, whereas the concentration of pe-
largonidin glucoside did not reach the level in wild-type plants.
The largest amounts of anthocyanins were found in immature
fruit of ANRi line 17. However, in later stages, the concentration
leveled out and was indistinguishable from the levels in the other
transgenic lines. Flavonols (B-ring-dihydroxylated quercetin and
monohydroxylated kaempferol) behaved differently. Whereas
quercetin glucuronide was strongly up-regulated in ANRi lines,
kaempferol glucoside was down-regulated (Fig. S6).
The concentration of monomeric catechin was measured at
various fruit ripening stages in untransformed control and ANRi
lines (Fig. 7). Catechin levels were reduced signicantly in ANRi
fruits of the late ripening stages in comparison with wild-type
fruits. By contrast, free epicatechin was not found in a quanti-
able concentration in any of the tissues analyzed.
Proanthocyanidins with a low DP can be analyzed directly by
LC-ESI-MS
n
and distinguished by their mass spectra and reten-
tion times. Here, four dimeric forms were found and quantied,
two stereoisomeric forms being two-fold B-ring dihydroxylated
(two (epi)catechin subunits) and two being mixed mono- and di-
hydroxylated ((epi)afzelechin with (epi)catechin subunit) (Fig. 8).
However, ANR products (epiafzelechin and epicatechin) cannot
be discriminated from those of LAR (afzelechin and catechin).
The levels of dimeric proanthocyanidins were affected differently
by the down-regulation of ANR (Fig. 8). Whereas (epi)catechin
(epi)catechin (isomer 2) and (epi)afzelechin(epi)catechin (iso-
mer 2) showed statistically signicantly reduced levels in fruits of
ANRi plants compared with controls at all developmental stages,
(epi)catechin(epi)catechin (isomer 1) displayed lower concentra-
tions mainly in later ripening stages. Minor changes were
observed for (epi)afzelechin(epi)catechin (isomer 1).
Analysis of higher proanthocyanidins by benzylthiol-
mediated thiolysis
Proanthocyanidins can be analyzed for their starter and chain a-
van-3-ol subunits by thiolysis with benzylthiol. In addition, DP
can be calculated from the relative amounts of all subunits to the
starter unit (Table 1). Comparison of the subunits released by
thiolysis showed that the ratio of LAR (catechins) to ANR (epi-
catechin, epiafzelechin) products increased in ANRi lines. In par-
ticular, the proportions of 3,4-trans-epicatechin and epiafzelechin
chain subunits decreased from 59.2% and 3.6% to 23.1% and
2.0%, respectively. By contrast, catechin starter and chain
Fig. 2 Gene expression analyses of avonoid genes in tissues of the strawberry (Fragaria 9ananassa) ANRi line F17 relative to wild-type control and
normalized by ITS (internally transcribed spacer of ribosomal DNA) expression. The normalized expression levels are shown on a logarithmic scale to
visualize the down-regulation (values < 1). Error bars, SD; signicance level according to t-test is given (***0.1%). ANR, anthocyanidin reductase; ANS,
anthocyanidin synthase; CHI, chalcone isomerase; C4H, cinnamic acid-4-hydroxylase; C4L, 4-coumarate:CoA ligase; CHS, chalcone synthase; DFR,
dihydroavonol-4-reductase; FGT1, UDP-glucose:avonoid-3-O-glucosyltransferase; FHT, avanon-3-b-hydroxylase (syn. F3H); FLS, avonol synthase;
ITS, internally transcribed spacer (rDNA); LAR, leucoanthocyanidin reductase; MYB, FaMYB1 transcription factor; PAL, phenylalanine ammonia-lyase.
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subunits increased from 16.0%, 3.0% and 17.1% to 25.0%,
26.7% and 21.3%, respectively. However, the percentage of epi-
catechin starter rose from 1.2% to 1.8%. The data show that
ANR products (epiafzelechin, epicatechin) are underrepresented
in the ANRi lines, whereas LAR products (catechin) are up-regu-
lated. The modication of the starter and chain subunits also
resulted in shorter proanthocyanidin chain lengths in the ANRi
lines, with a mean chain length of 3.8 0.4 avan-3-ol units per
chain, in comparison with the untransformed control, with a
chain length of 5.8 0.1 (Table 1).
Pollen viability
From petunia and maize mutants, it is known that avonols are
essential in these systems for pollen fertility. Flavonol-decient
mutants are self-sterile (Mo et al., 1992). The strongly increased
level of quercetin glucuronide in fruit tissue (data not shown) of
the ANRi lines suggested experimental testing of pollen viability,
and some other ower traits (Table 2).
Pollen viability increased in ANRi lines (F14, F15, F21, F22)
in comparison with the wild-type plant Senga Sengana from
42.7% to 72.5%, but other ower traits, such as the number of
petals, sepals and anthers, were essentially unchanged, except for
the faint anthocyanin color of older petals.
Discussion
Transgenic lines
In the present study, 13 transgenic strawberry lines of
F. 9ananassa cv Senga Sengana were successfully established
using the transformation vector pBI-ANRi. The transgenic lines
originate from independent transformation events and contain
between one and six T-DNA copies. Each line was successfully
Fig. 3 Anthocyanin phenotype of strawberry (Fragaria 9ananassa) ANRi line F17 in comparison with the untransformed control. Top left, ower
phenotype (left, untransformed control; right, ANRi line); top right, root phenotype (left, untransformed control; right, ANRi line); middle series, fruit
phenotypes of untransformed control; bottom series, fruit phenotypes of ANRi line; G12, green fruit stages; W, white fruit stage; T, turning red stage;
R, red stage. Bars, 1 cm.
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propagated in vitro and transferred to the glasshouse. Glasshouse
plants developed normally. No developmental differences were
obvious compared with the wild-type, except for red coloration
of some transgenic plant tissues.
ANR down-regulation impacts on proanthocyanidin
formation
The analysis of transformed lines for integration events and gene
expression analysis demonstrated that ANR was down-regulated
in a number of independent transformed strawberry lines
(Fig. 2). The direct products of the down-regulated enzyme are
epicatechin and epiafzelechin (Fig. 1). In histochemical analyses
with DMACA, their staining is overlain by catechin/afzelechin
biosynthesized by LAR, as the reagent cannot discriminate ste-
reochemically different groups of avan- 3-ols (Fig. 1). However,
the occurrence of polymers with both types of monomer strongly
argues for identical patterns of epicatechin and catechin expres-
sion. The anthocyanin pattern was identical to the proanthocy-
anidin pattern with its expected allover epicatechin component.
LC-MS analysis showed signicantly reduced levels of mono-
meric catechin in fruit of ANRi lines, whereas free epicatechin
could not be found in strawberry fruit (Fig. 7). Subunit analysis
of polymeric proanthocyanidins from ANRi lines revealed higher
incorporation rates of catechin at the expense of epicatechin
units, probably owing to the reduced levels of epicatechin caused
by ANR down-regulation (Table 1). The increased ow of cate-
chin and/or its precursors into polymeric proanthocyanidins can
explain the small amounts of monomeric catechin in the trans-
genic lines (Fig. 1). Similarly, levels of dimeric proanthocyanins
composed of (epi)catechins were reduced in ANRi lines when
compared with controls (Fig. 8). However, the content of the
starter molecule epicatechin in polymeric anthocyanins was
almost unaffected (Table 1). The proportion of epicatechin as
starter increased slightly from 1.2% in the control, by a factor of
1.50, to 1.8% in ANRi lines. Likewise, the content of the starter
molecule catechin rose from 16.0%, by a factor of 1.56, to
25.0%. The total gain in starter molecules in comparison with
chain (extension) units results from the reduced chain length of
polymeric proanthocyanidins, which decreased from 5.8 units in
the control, by a factor of 1.52, to 3.8 units in ANRi lines. How-
ever, major changes were observed for the composition of the
extension units. Whereas, in wild-type plants, the epicatechin
chain subunits accounted for 62.8%, this value decreased to
25.1% in the ANRi lines. Thus, although the total production of
epicatechin is strongly reduced in the transgenic lines, as indi-
cated by the content of the extension units, the ratio of the cate-
chin/epicatechin starter molecules remained almost unchanged
at 13.3 (control) and 13.8 (ANRi). This points to an ANR-inde-
pendent formation of the starter molecules.
ANR down-regulation induces premature and ectopic
anthocyanin formation
The prominent effect on the other avonoid classes, such as
anthocyanins (ANS products) and avonols (avonol synthase
(FLS) products) can mostly be explained by the articial bottle-
neck causing higher substrate concentration for these diverging
biosynthetic steps (Fig. 1). The observed effect of premature and
ectopic anthocyanin formation in strawberry fruit and stigmata,
respectively (Fig. 3), also demonstrates that the enzymes required
for glycosylation of anthocyanidins are already present in early
strawberry fruit development and stigmata, or that other gly-
cosylating enzymes with lesser substrate specicity can take over
when the ratio of competing enzyme activity changes by experi-
mental down-regulation of ANR. A UDP-glucose:anthocyanidin
3-O-glucosyltransferase (FaGT1) and two avonol glucos-
yltransferases (FaGT6 and 7) have been characterized from
F. 9ananassa (Almeida et al., 2007; Griesser et al., 2008a,b).
They show distinct, but overlapping, enzymatic activities and
spatiotemporal expression patterns. FaGT1, which adds glucose
moieties to pelargonidin and cyanidin, is weakly expressed in
Wild type
ANRi 17
No staining DMACA staining
Fig. 4 Anthocyanin phenotype (red stigmata) of owers and receptacle as
well as histochemical analysis by dimethylaminocinnamicaldehyde
(DMACA) staining for (epi)catechinsproanthocyanidins of strawberry
(Fragaria 9ananassa) ANRi line F17 in comparison with untransformed
control. Top, ower and receptacle of untransformed control; bottom,
ower and receptacle of ANRi line; left, visible (anthocyanin) phenotype;
right, DMACA staining for catechinsproanthocyanins. Bar, 1 cm.
Fig. 5 Histochemical analyses for avonols in strawberry
(Fragaria 9ananassa) fruit with diphenylboric acid 2-aminoethylester
(DPBA)-UV. Left, untransformed control; right, ANRi line F17. Bar, 1 cm.
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immature strawberry fruit, but its transcript levels increase signif-
icantly during ripening (Almeida et al., 2007; Griesser et al.,
2008b). A glucuronyltransferase has not yet been described from
Fragaria. Thus, the enzyme involved in the accumulation of
quercetin glucuronide, which increased signicantly in the ANRi
line probably as a result of the higher availability of substrates,
remains unknown.
Most prominent in the ANRi lines is the visible anthocyanin
phenotype in all organs which form epicatechin/epiafzelechin in
wild-type strawberries. These are mainly unripe fruits, stigmata
of owers and root tips (Hoffmann et al., 2012). As the down-
regulation of ANRi led to the accumulation of anthocyanins in
these tissues, it appears that UDP-glucose:avonoid-3-O-gluco-
syltransferase (FGT1) and ANR compete for their common sub-
strates pelargonidin and cyanidin. This is accompanied by the
depletion of leucoanthocyanidin substrate supply for ANS by
strong LAR activity, producing high levels of catechin and afzele-
chin in unripe fruits. Similarly, the down-regulation of FaGT1 in
F. 9ananassa caused both a reduction in anthocyanin pigments
and accumulation of signicant levels of epiafzelechin synthe-
sized by ANR from pelargonidin in fruits of the transgenic lines
(Griesser et al., 2008b).
Secondary effects in ANRi lines
The secondary effects of ANR down-regulation were ambigu-
ous for avonols. The concentration of the main avonol
quercetin glucuronide rose strongly according to histochemical
analysis (Fig. 5) and analytical quantication by LC-MS analy-
sis (Fig. S5). The minor avonol kaempferol glucoside, which
reached only a 10-fold lower amount in strawberry fruits in
comparison with quercetin glucuronide, showed reduced levels
in ANRi lines. Quercetin glucuronide is probably increased in
ANRi lines because of the accumulation of upstream avonoid
metabolites. By contrast, lower levels of kaempferol gluco-
side may be caused by depletion of the precursor UDP-glucose
by competing consumption by the induced anthocyanin
formation (Fig. 1).
The observed higher pollen viability of ANRi lines argues for a
role of avonols in pollen fertility in strawberry, as described for
petunia and maize (Mo et al., 1992; Taylor & Jorgensen, 1992).
Chalcone synthase-decient petunia plants produced abnormal
anthers devoid of avonoid pigments. Although viable pollen was
produced, pollen germination and tube growth were severely
reduced, indicating an effect of avonoids on pollen fertility.
(a)
(b)
Fig. 6 Relative concentrations of cyanidin
glucoside (a) and pelargonidin glucoside (b)
in relation to an internal standard (4-
methylumbelliferyl glucoside) in strawberry
(Fragaria 9ananassa) fruits of different ANRi
lines (F22, F21, F17, F15 and F14) and the
wild-type (wt). The mean values + SD were
calculated and plotted. Statistical signicance
was calculated with the WilcoxonMann
Whitney U-test and marked with asterisks
(*, P 0.05; **, P 0.01). G12, green fruit
stages (G1, green receptaculum tissue
between achenes; G2, late green stage); W,
white fruit stage; T, turning red stage; R, red
stage.
Fig. 7 Relative concentrations of catechin in relation to an internal standard (4-methylumbelliferyl glucoside) in strawberry (Fragaria 9ananassa) fruits of
different ANRi lines (F22, F21, F17, F15 and F14) and the wild-type (wt). The mean values + SD were calculated and plotted. Statistical signicance was
calculated with the WilcoxonMannWhitney U-test and marked with asterisks (*, P 0.05; **, P 0.01). G12, green fruit stages; W, white fruit stage;
T, turning red stage; R, red stage.
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ANR down-regulation does not seem to affect fruit rmness
negatively, but it does so for ascorbic acid content (Table S2).
Ascorbic acid is a cofactor of 2-oxoglutarate-dependent dioxygen-
ases which are also involved in avonoid biosynthesis (avanon-
3-b-hydroxylase (FHT, syn. F3H), FLS, ANS) (Halbwirth et al.,
2006). Fruit rmness may be inuenced by polyphenols which
are integrated into secondary cell walls: these may be avan-3-ols,
but also hydroxycinnamic acids, diverging before the committed
steps of avonoid biosynthesis (Braidot et al., 2008).
Statistically relevant changes have also been found at the gene
expression level for avonoid genes, with increases or decreases
by factors of up to ten in various tissues. Such secondary effects
on gene expression have also been observed by experimental
down-regulation of other avonoid genes in other systems, such
Fig. 8 Relative concentrations of dimeric
proanthocyanidins in relation to an internal
standard (4-methylumbelliferyl glucoside) in
strawberry (Fragaria 9ananassa) fruits of
different ANRi lines (F22, F21, F17, F15
and F14) and the wild-type (wt). The
mean values + SD were calculated and
plotted. Statistical signicance was
calculated with the WilcoxonMann
Whitney U-test and marked with asterisks
(*, P 0.05; **, P 0.01). G12, green fruit
stages; W, white fruit stage; T, turning red
stage; R, red stage.
Table 1 Concentration (%) of proanthocyanidin starter and chain subunits in three independent ANRi lines of strawberry (Fragaria 9ananassa) and
wild-type control (mean values from four technical repetitions each) by benzylthiol thiolysis and subsequent analytical LC separation
ANR products LAR products
Chain length
Starter
Chain subunit
Starter
Chain subunit
Epicatechin
3,4-trans-Epicatechin
benzylthioether
Epiafzelechin
benzylthioether Catechin
3,4-trans-Catechin
benzylthioether
3,4-cis-Catechin
benzylthioether
Control 1.2 0.1 59.2 0.7 3.6 0.2 16.0 0.5 3.0 0.1 17.1 0.7 5.8 0.1
ANRi lines 1.8 0.5 23.1 6.5 2.0 1.3 25.0 3.0 26.7 7.2 21.3 3.9 3.8 0.4
Starter units result in free catechins by thiolysis; chain subunits are found as 4-benzylthioethers. The biosynthetic enzyme (either anthocyanidin reductase
(ANR) or leucoanthocyanidin reductase (LAR)) for the respective type of subunit is indicated. Chain length (mean number of avan-3-ol units per chain)
was calculated from the relative amounts of all subunits to the starter unit.
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as the silencing of ANS in apple (Malus 9domestica) (Szankow-
ski et al., 2009) and avonol glucosyltransferase in Arabidopsis
thaliana (Yin et al., 2012). Interestingly, such gene expression
changes are also observed by experimental manipulation of
avonoid levels with an enzyme inhibitor acting on 2-oxogluta-
rate-dependent dioxygenases involved in avonoid biosynthesis
(Fischer et al., 2006). Taken together, this strongly argues for the
existence of feedback regulation mechanisms which regulate a-
vonoid gene expression via the sensing of avonoid biosynthesis
precursors, intermediates or end products.
Conclusion
Experimental down-regulation of ANR, which catalyzes a com-
peting step to anthocyanin formation, led to a prominent antho-
cyanin phenotype with ectopic and premature red coloration in
Fragaria 9ananassa. Epicatechin, the direct product of ANR,
was consequently down-regulated as a chain extension unit in the
polymeric proanthocyanidins. However, as a starter unit of pro-
anthocyanidins, the epicatechin proportion was found to remain
unchanged, supporting the view of different epicatechin pools for
starter and chain subunits. An increasing concentration of the a-
vonol quercetin glucuronide was interpreted as a secondary effect
via the accumulation of intermediates and higher substrate avail-
ability for this biosynthesis. Effects on gene expression of other
avonoid genes were also observed and could be explained by
feedback regulation via avonoid end products, intermediates or
precursors of avonoid biosynthesis.
Acknowledgements
The Bayerisches Staatsministerium f ur Umwelt und Gesundheit
is gratefully acknowledged for supporting the Rosaceae rhizo-
sphere project. Elizabeth Schroeder-Reiter (Ludwig-Maximilians-
University Munich) is acknowledged for proof-reading of the
manuscript as a native speaker. The authors acknowledge
I. Hiller, U. Hille, I. Polster and G. Klotzsche for technical
assistance.
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Table 2 Test for pollen viability and other ower-related traits with ANRi lines of strawberry (Fragaria 9ananassa) and wild-type control
Line n
Number of
petals
Number of
sepals
Number of
anthers
Color of the
receptacle
Test on pollen viability
Number of counted
pollen grains
Percentage of
vital pollen grains
F14 10 11.4 1.58 5.8 0.92 24.9 2.33 Red 374 72.5
F15 10 11.2 1.14 5.6 0.70 25.2 3.91 Red 329 62.9
F21 10 11.4 1.58 5.6 0.84 27.2 3.65 Red 216 68.5
F22 10 10.9 1.45 5.5 0.85 23.7 3.27 Red na na
Wild-type 10 11.2 1.93 5.9 0.99 27.3 3.09 Green 255 42.7
na, not available.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Construction of vector pBI-ANRi.
Fig. S2 PCR-based detection of the nptII selectable marker gene
in 14 of 17 transgenic strawberry lines.
Fig. S3 PCR-based detection of the chimeric anr gene construct
in 14 of 17 transgenic strawberry lines.
Fig. S4 Detection of integrated T-DNA copies in DNA of the
ANRi transgenic strawberry lines by Southern hybridization.
Fig. S5 Gene expression analyses of avonoid genes in receptacles
of ve independent strawberry ANRi lines (F22, F21, F17, F15
and F14) relative to wild-type control.
Fig. S6 Relative concentration of quercetin glucuronide (top)
and kaempferol glucoside (bottom) in fruits of different ANRi
lines (F22, F21, F17, F15 and F14) and the wild-type.
Table S1 Gene-specic primers: MYB, PAL, C4H, C4L, CHS,
CHI, F3H, DFR, FLS, ANS, LAR, ANR, FGT1 and ITS (internal
transcribed spacer 26S-18S RNA, housekeeping transcript for
normalization)
Table S2 Tests for fruit rmness and ascorbic acid concentration
in ANRi lines of strawberry
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