Protocolo de Antocianinas 2

Premature and ectopic anthocyanin formation by silencing ofanthocyanidin reductase in strawberry (Fragaria9 ananassa)

Thilo C. Fischer1, Beate Mirbeth1, Judith Rentsch2, Corina Sutter3, Ludwig Ring3, Henryk Flachowsky2,

Ruth Habegger3, Thomas Hoffmann3, Magda-Viola Hanke2 and Wilfried Schwab3

1Plant Biochemistry and Physiology, Ludwig-Maximilians-University Munich, Großhadernerstr 2–4, D-82152, Planegg-Martinsried, Germany; 2Julius K€uhn-Institute – Federal Research

Centre for Cultivated Plants, Institute for Breeding Research on Horticultural and Fruit Crops, Pillnitzer Platz 3a, 01326 Pillnitz, Dresden, Germany; 3Biotechnology 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 74754Email: thilo.fischer@biologie.

uni-muenchen.de

Wilfried Schwab

Tel: +49 8161 71 74754

Email: [email protected]

Received: 10 June 2013Accepted: 19 August 2013

New Phytologist (2013)doi: 10.1111/nph.12528

Key words: anthocyanidin reductase (ANR),anthocyanin, epicatechin, flavonols,proanthocyanidins, RNAi, strawberry(Fragaria9 ananassa).

Summary

� Strawberry (Fragaria9 ananassa) is a fruit crop with a distinct biphasic flavonoid 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 flowers, 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 flavonol

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 (Fragaria9 ananassa), flavonoids are secondarymetabolites that are of interest because of their multiple biologi-cal functions in plants as well as in human consumption. In ripefruits, the bright red anthocyanin pelargonidin and, to a lesserdegree, the dark red cyanidin (dependent on cultivar) are decisivefor fruit attractiveness. Flavonols are also present and theirhealth-promoting effects have been broadly discussed (Graf et al.,2005; Munoz et al., 2011). Monomeric and polymeric flavan-3-ols mainly occur in unripe fruits (reviewed in Flachowsky et al.,2011). The major flavonoid genes of strawberry have beencloned, characterized with respect to general substrate specificityand the gene copy numbers of the octoploid strawberry have beenstudied (Almeida et al., 2007) (Fig. 1). For some genes and theirrespective enzymes, more specific functions have been studied,such as for flavonol-O-glucosyltransferases (Griesser et al., 2008a)and dihydroflavonol-4-reductase/flavanone-4-reductase (Fischer

et al., 2003). In addition, the genome of the diploid woodlandstrawberry Fragaria vesca has been elucidated (Shulaev et al.,2011), providing a broad base of information on orthologousand paralogous genes from one of the precursors of cultivatedoctoploid strawberry.

Flavan-3-ols are a diverse class of flavonoids. Monomericflavan-3-ols (catechins in general) differ in stereochemistry at theC3 hydroxyl group in the C-ring. 2,3-trans-Catechin (catechin inthe strict sense) is formed via the reduction of leucocyanidin byleucoanthocyanidin-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 the3′,4′-dihydroxylated flavan-3-ols catechin and epicatechin,whereas the 4′-monohydroxylated equivalents are afzelechin andepiafzelechin, which are also formed in strawberry. By contrast,

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the 3′,4′,5′-trihydroxylated gallocatechin and epigallocatechin arenot known from strawberry. Proanthocyanidins are polymericflavan-3-ols and are supposed to be formed from such mono-meric building blocks. Starter and extension units are connectedby an as yet unknown mechanism. Almost no free epicatechin isfound in strawberry, but it is quite common as a subunit in theproanthocyanidins (Buend�ıa et al., 2010). Proanthocyanidinsshow much structural variation with respect to the kinds of build-ing blocks (C3-hydroxy group stereochemistry and B-ringhydroxylation), chain length and stereochemistry of the interflav-anoid bond. Specific biological effects may be expected, but theiranalysis is hampered by demanding analytics and difficultpreparative isolation.

Experimental manipulation of flavan-3-ol metabolism hasbeen performed with a number of systems. Mutation, silencingand overexpression of transcription factors (tt2, tt8, ttg1) havebeen performed to identify specific functions with the systemArabidopsis thaliana (Debeaujon et al., 2003). For the Rosaceaefamily member apple (Malus9 domestica), which is related tostrawberry, Myb-type transcription factors for (pro)anthocyaninbiosynthesis have been identified (Takos et al., 2006; Ban et al.,2007; Espley et al., 2007), whereas, in strawberry, the factorFaMYB1 has been shown to control anthocyanin biosynthesis(Aharoni et al., 2001) and to suppress anthocyanin and toenhance flavan-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 toenhanced levels of specific proanthocyanidins of up to 100-fold(Li et al., 2007; Flachowsky et al., 2009). The last step before thefinal branching point between epicatechin and anthocyanin bio-synthesis is catalyzed by anthocyanidin synthase (ANS), which isresponsible for anthocyanidin formation. Subsequently, flavo-noid-3O-glycosyltransferases (FGTs) or ANR performs the com-mitted steps towards either stable anthocyanin or epicatechin andits polymers. ANR has been overexpressed in Arabidopsis andheterologous systems (Nicotiana) to demonstrate its function(Xie et al., 2003). The structural genes have also been targets forsilencing approaches. In apple, the ANS gene ans has beensilenced, resulting in the expected abolishment of anthocyaninformation, but this also led to an unexpected rise in free epicate-chin (Szankowski et al., 2009). In strawberry, redirection of theanthocyanin pathway to flavan-3-ols was observed by the down-regulation of an anthocyanidin glucosyltransferase in ripeningstrawberry fruit (Griesser et al., 2008b).

In this work, experimental manipulation in strawberry wasperformed by the silencing of ANR. In strawberry, this is at thedevelopmental switch between early proanthocyanidins, whichmight be involved in pathogen defense (Jersch et al., 1989), andlate color-determining anthocyanins. This approach resulted instrong unbalancing of flavonoid classes and a striking anthocya-nin phenotype in unripe fruits. Ectopic anthocyanin formationin other epicatechin-forming organs (Hoffmann et al., 2012),such as the stigmata of flowers and root tips, was also observed.

Materials and Methods

Plant material

The octoploid strawberry (Fragaria9 ananassa Duch.) cv SengaSengana was used for this study. For convenience, the unripe andripe receptacles of strawberry are referred to as ‘fruit’ in thisarticle, even though they are not botanically defined as such.

Preparation of silencing intron-hairpin vector construct

The pBI-ANRi construct was generated according to Hoffmannet al. (2006). b-Glucuronidase (GUS) of pBI121 (Jefferson,1987) was replaced by the second intron of the F.9 ananassaenone oxidoreductase gene (AY158836, nucleotides 4886–4993).A 115-bp fragment corresponding to nucleotides 32–146 ofF.9 ananassa ANR mRNA (DQ664192) was inserted into the 5′and 3′ arms of the intron. This ANR gene had been identified bycDNA cloning (Almeida et al., 2007) and represents the majorexpressed gene of ANR in octaploid strawberry. The resultingplasmid 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 shootproliferation medium containing Murashige and Skoog (MS)

Fig. 1 Biosynthesis of flavonoids in strawberry (Fragaria9 ananassa). Onlydihydroxylated flavonoids and precursors are shown; biosynthesis ofmonohydroxylated flavonoids occurs in parallel. ANR, anthocyanidinreductase; ANS, anthocyanidin synthase; CHI, chalcone isomerase; C4H,cinnamic acid-4-hydroxylase; C4L, 4-coumarate:CoA ligase; CHS,chalcone synthase; DFR, dihydroflavonol-4-reductase; FGT1, UDP-glucose:flavonoid-3-O-glucosyltransferase; FHT, flavanon-3-b-hydroxylase (syn. F3H); FLS, flavonol synthase; LAR, leucoanthocyanidinreductase; PAL, phenylalanine ammonia-lyase. Changes caused bysilencing of anthocyanidin reductase ANR are also indicated by arrows(see the Results section).

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salts and vitamins (Murashige & Skoog, 1962) supplementedwith 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% DifcoBacto-agar (Difco, Heidelberg, Germany). Plants were grownin a culture chamber (16 h light at 21°C and 8 h dark at16°C) and subcultured every 3–4 wk. A single colony of theAgrobacterium tumefaciens strain AGL0 containing the plasmidvector pBI-ANRi was grown overnight in 2 ml of liquid Luria–Bertani (LB) medium supplemented with 100 lg l�1 rifampicinand 100 lg l�1 kanamycin. LB plates were inoculated bystreaking 30 ll of the liquid cultures. Five plates were incu-bated for 4 d at 28°C. The bacteria were resuspended in 25 mlof simplified induction medium (SIM) (2% sucrose, 20 mMsodium citrate, 0.1 mM acetosyringone, 1 mM betaine hydro-chloride, pH 5.2), and grown at 28°C and 220 rpm on a hori-zontal shaker to give a final optical density at 600 nm (OD600)of 0.8–1. Plant material was dipped into this bacterial suspen-sion and was subsequently co-cultivated in the dark at 25°Con solid regeneration medium consisting of 4.4 g l�1 MS saltsand vitamins (M0222; Duchefa, RV Haarlem, theNetherlands), 2% sucrose, 9 mM thidiazuron (TDZ), 0.9 mMIAA and 0.3% gelrite at pH 5.7 for 3 d. Leaf explants werewashed 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 H2O for 5 min and, finally, in MSO again asdescribed. Adventitious shoot induction was performed onregeneration medium supplemented with 500 mg l�1 timetinand 300 mg l�1 kanamycin. Explants were cultured in the darkfor 3 wk at 25°C, and then under a 16 h : 8 h photoperiod atthe same temperature. Regenerated meristems were excised 9–12 wk after inoculation and independent transgenic clonesfrom a single transformation event were obtained. Transgenicshoots were subcultured on shoot proliferation medium with500 mg l�1 timetin and 300 mg l�1 kanamycin under a photo-period of 16 h of light at 21°C and 8 h of darkness at 16°C.Rooted plantlets were transferred into 5-cm plastic pots withsoil and acclimatized in the glasshouse. Independent T0 lineswere propagated via stolons to provide sufficient plant materialfor the studies.

PCR and Southern blot analysis

Genomic DNA was extracted from in vitro leaves using a modi-fied cetyltrimethylammonium bromide (CTAB) extraction pro-tocol. PCRs for testing the transgenic plants for the presence oftransgenic DNA were performed in 25 ll of reaction mixturecontaining 50 ng DNA, 19DreamTaqTM buffer, 0.2 mM deoxy-nucleoside triphosphates (dNTPs), 0.5 lM of each primerand 0.5 U DreamTaqTM DNA polymerase (MBI Fermentas, St.Leon-Roth, Germany). The PCR started by initial denaturationat 94°C for 4 min, followed by 33 cycles of 30 s of denaturationat 94°C, 1 min of annealing at 56°C and 1.5 min of extension at72°C, and a final extension at 72°C for 7 min. All PCRs wereperformed in a MyCyclerTM thermocycler (Bio-Rad, Hercules,CA, USA). The PCR for detection of the nptII marker gene wasperformed using the primers nptIIF (5′-ACAAGATGGATTG

CACGCAGG-3′) and nptIIR (5′-AACTCGTCAAGAAGGCGATAG-3′), whereas, for the chimeric ANR gene, the primersANRF (5′-CGCACAATCCCACTATCCTT-3′) and ANRR(5′-TCGCCCTTAAGTCTGCTTGT-3′) were used.

Southern hybridization experiments were performed using10 lg of HindIII (MBI Fermentas)-digested DNA. The DNAwas digested with 100 U of HindIII at 37°C overnight. Therestricted DNA was separated on a 0.8% agarose gel and trans-ferred onto a nylon membrane (Roche Diagnostics, Mannheim,Germany). PCR-amplified, digoxigenin-labeled probes from thecoding region of the nptII marker gene were generated using theprimers nptIIF/R and used for hybridization. Hybridization anddetection were performed using the ECF Random Prime Label-ing and Detection Kit (Amersham Biosciences, Freiburg,Germany) according to the manufacturer’s manual.

Histochemical analysis

Dimethylaminocinnamicaldehyde (DMACA) staining for flavan-3-ols (Feucht reaction) of tissues was performed by exposure for1 min in 1% DMACA (w/v) in ethanol/6M HCl (1 : 1). Ethanolwas used instead of n-butanol (W. Feucht, pers. comm.). Thespecificity of the assay has been studied previously (Treutter,1989; Hoffmann et al., 2012).

For diphenylboric acid 2-aminoethylester (DPBA) staining forflavonols, plant tissues were vacuum infiltrated with a saturatedDPBA solution of 0.5% w/v DPBA (Sigma-Aldrich) and 0.1%Triton X-100 for 30 min. Subsequently, the color was detectedunder UV long-wavelength light (UV lamp, type 600352;Waldmann, Villingen-Schwenningen, Germany). Photographswere taken using a digital camera (D40X; Nikon, Dusseldorf,Germany).

Gene expression analysis

Transcript levels of specific and general flavonoid pathway geneswere analyzed in different tissues and developmental stages. Forflower analysis, petals, stigmata and receptacles were separatedand frozen in liquid nitrogen. Fruits were separated from greenplant material (leaves, stem) and also frozen in liquid nitrogen.Fruits at the stages ‘early green’ (G1) (achenes clearly separatedby green tissue of receptaculum) and ‘early white’ (W) (startingto lose green color and becoming white) were harvested. FromANRi 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 manufacturer’s protocol. Except forpetals, which were lyzed by buffer RLC, all samples were lyzed bybuffer RLT. Both buffers contained b-mercaptoethanol. RNAwas diluted in 30 ll (stigma, petals), 50 ll (receptacle, greenfruit) or 100 ll (white fruit) RNAse-free water. The RNA wasapplied directly to quantitative PCR (qPCR) after proof of thepurity of RNA with respect to DNA (data not shown). cDNAsynthesis was performed according to recommended conditionsfor Superscript reverse transcriptase (Bio-Rad). Random hex-amer primers (New England Biolabs, Ipswich, MA, USA) and


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20–50 ng of total RNA were used. qPCR was performed with aBio-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-specific 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 whitefruit) or 6.3 ll (white fruit) of water. Mastermixes were used toavoid inaccuracies. Every type of reaction was performed at leastthree times. The PCR program involved initially 3 min at 95°C,followed by 40 cycles (55 cycles for white fruit analyses) of 10 s at95°C, 30 s at 55°C and 10 s at 72°C. Primer sequences are givenin Table S1. The relative expression ratio was calculated and nor-malized according to Pfaffl (2001).

Liquid chromatography-electrospray ionization-massspectrometryn (LC-ESI-MSn) analysis

A Bruker Daltonics esquire 3000plus ion trap mass spectrometer(Bruker Daltonics, Bremen, Germany) connected to an Agilent1100 HPLC system (Agilent Technologies, Waldbronn,Germany), equipped with a quaternary pump and a diode arraydetector, was utilized for metabolite analysis. Components wereseparated with a Phenomenex Luna C-18 column (150 mmlong9 2.0 mm i.d., particle size 5 lm; Phenomenex, Aschaffen-burg, Germany) that was held at 28°C. High-performance liquidchromatography (HPLC) was performed with the followingbinary gradient system: solvent A, water with 0.1% formic acid;solvent B, 100% methanol with 0.1% formic acid. The gradientprogram was as follows: 0–30 min, 100% A to 50% A/50% B;30–35 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 flow rate was0.2 ml min�1. The ionization parameters were as follows: thevoltage of the capillary was 4000 V and the end plate was set to�500 V. The capillary exit was 121 V and the Octopole RFamplitude was 150 Vpp (peak-to-peak voltage). The temperatureof the dry gas (N2) was 330°C at a flow rate of 9 l min�1. Thefull scan mass spectra of the metabolites were measured at m/z50–800 until the ICC target reached either 20 000 or 200 ms.Tandem MS was carried out using helium as the collision gas(3.569 10�6 mbar) with a collision voltage of 1 V. Mass spectrawere 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 differentcompounds. Samples (fruit of different developmental stages,roots, leaves) were individually frozen, lyophilized for 48 h andhomogenized with a mill (Retsch MM 200, Haan, Germany) toa fine powder. An aliquot of 50 mg of lyophilized powder wasused for each of the three biological replicates. Non-natural4-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 llmethanol, vortexing and sonication for 10 min, the sample wascentrifuged at 16 000 g for 10 min. The supernatant was removedand the residue was re-extracted with 500 ll of methanol. Thesupernatants 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 centrifugationat 16 000 g, the clear supernatant was used for LC-MS analysis.Each extract was injected twice (technical replicate). Metaboliteswere identified by their retention times, mass spectra and production spectra in comparison with the data determined for authen-tic reference material. Phenylpropanoyl glucose esters wereenzymatically synthesized with FaGT2 (Fragaria 9 ananassaglucosyltransferase 2) (Lunkenbein et al., 2006). Pelargonidin3-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 identified accord-ing to Fossen et al. (2004). Statistical evaluation was performedusing independent Student’s t-test of SOFA Statistics (Paton-Simpson & Associates Ltd, Auckland, New Zealand). The majorknown phenolic metabolites were quantified in the positive(anthocyanins) and negative (phenylpropanoids, flavonoids) MSmode by the IS method and were expressed as & equivalent ofdry weight assuming a response factor of 1. The metaboliteconcentrations did not always lie within the linear range of thedetector and the calculation of their relative levels did not allowimmediate comparison with the absolute levels of phenolics pro-vided by other studies, but the method offered the advantage toobtain relative values in a short period of time which is sufficientto compare relative metabolite levels. Signals of the compoundswere integrated in their [M +H]+, [M –H]� or [M +HCOO]�

ion traces. The non-natural 4-methylumbelliferyl glucoside wasused as IS for the relative quantification of the metabolites.Moreover, this compound served as a control for the ionizationyield 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 metabolitequantification was performed using the DataAnalysis 3.1 andQuantAnalysis 1.5 software (Bruker Daltonics), normalizing allresults to the IS.

Analysis of higher proanthocyanidins by benzylthiolthiolysis

Thiolytic degradation and degree of polymerization (DP) calcula-tions were performed according to Gu et al. (2003). Degradationproducts were analyzed by high-performance liquid chromatogra-phy-diode array detection-electrospray ionization-mass spectrom-etryn (HPLC-DAD-ESI-MSn) consisting of a Bruker Daltonicsesquire 3000plus 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-18column (150 mm long9 2.00 mm i.d., particle size 5 lm; Phe-nomenex) that was held at 28°C. HPLC was performed with thefollowing binary gradient system and gradient: solvent A, waterwith 0.1% formic acid; solvent B, methanol with 0.1% formicacid; 0–30 min, 100% A to 50% A/50% B; 30–35 min, 50% A/50% B to 100% B, hold for 15 min; 100% B to 100% A in5 min, then hold for 10 min. The ESI voltage of the capillary was



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set to �4000 V and the end plate to �500 V. Nitrogen was usedas dry gas at a temperature of 330°C and a flow rate of 9 l min�1.The capillary exit was 121 V and the Octopole RF amplitude was150 Vpp. Tandem MS was carried out using helium as the colli-sion gas (3.569 10�6 mbar) with a collision voltage of 1 V. Spec-tra were acquired in positive and negative ionization mode.Components were identified by their retention times, mass spec-tra and product ion spectra in comparison with data determinedfor authentic reference materials. Quantification was performedby UV detection at 280 nm.

Ascorbic acid content

Ascorbic acid was quantified with the reflectometer RQflex® 10Reflectoquant® (Merck KGaA, Darmstadt, Germany) accordingto the protocol described by Pantelidis et al. (2007) with minormodifications. Fruit samples of 200 g were mixed with 200 ml ofoxalic acid (1%), homogenized for 1 min and centrifuged for3 min at 1500 g; 2.5 ml of the supernatant were mixed with2.5 ml of oxalic acid (1%) and supplemented with 250 mg ofpolyvinylpolypyrrolidone (PVP) to remove phenols, and threedrops of H2SO4 (25%) to reduce the pH ≤ 1. Results wereexpressed as milligrams of ascorbic acid per 100 ml of the super-natant supplemented with PVP and H2SO4.

Pollen viability test

The evaluation of pollen viability was performed as described byFlachowsky et al. (2007). Anthers of glasshouse flowers were col-lected, air dried for 3 d at room temperature and stored at�80°C. The number of vital and dead pollen grains was countedafter incubation in 0.5% carmine–acetic acid. Dead pollen grainsstay uncolored; vital pollen grains are red–orange.

Determination of fruit firmness

Firmness was recorded in three independent experiments, eachwith 7–22 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 fruitssqueezed from 100 to 250 g. A value of 200 g mm�1 indicatesthat 200 g of force will squeeze the fruit by 1 mm. The datawere 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 vectorpBI121. Such hairpin constructs give rise to the production ofsmall interfering RNAs (siRNAs) in plants. Three transforma-tion experiments were performed with the strawberry (Fragaria9 ananassa) cv ‘Senga Sengana’. A total of 530 wounded leaf

discs of ‘Senga Sengana’ was inoculated with the A. tumefaciensstrain AGL0 carrying the binary plasmid vector pBI-ANRi.The transformation experiments resulted in a total of 34 puta-tive transgenic plants representing independent transformationevents, which were selected after regeneration on regenerationmedium containing 500 mg l�1 timetin and 300 mg l�1 kana-mycin. Seventeen of these plants survived, and the first 14plants 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 primersnptII_F/R for nptII and ANR_F/R for the chimeric anr genesequence. All transgenic plants showed PCR products of theexpected sizes (Figs S2, S3). Transgene integration was testedby Southern hybridization (Fig. S4). All lines showed hybrid-ization products, except for line F19, which was excluded fromfurther investigations as a result of the contradictory resultsobtained by PCR and Southern hybridization.

Gene expression analysis

Transcript levels of flavonoid genes were analyzed by reverse tran-scription-quantitative polymerase chain reaction (RT-qPCR) indifferent tissues of the ANRi lines obtained from independenttransformation events. Comparison of the gene expression dataof wild-type and ANRi transgenic lines confirmed the down-regu-lation of the ANR gene, as indicated by small normalized expres-sion levels in various tissues, except petals. However, mRNAlevels of MYB, C4L, CHS, DFR, ANS and FGT appeared to beincreased significantly by a factor of up to 10 in certain tissues,and FaMYB1, PAL, C4L, CHI, DFR, FLS and FGT levels werereduced in some other tissues (Fig. 2). The down-regulation ofANR is also consistent for the independent transformation eventsanalyzed (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 transgenicplants. Line F17, which shows the strongest anthocyanin pheno-type, was chosen for further in-depth analysis.

Histochemical analysis

DMACA staining for (epi)catechins–proanthocyanidins allowedhistochemical analysis to compare the anthocyanin phenotypewith tissue-specific (epi)catechin–proanthocyanidin formation. Aco-localization of (epi)catechin–proanthocyanidin formation inthe untransformed control (stigmata and cortex) and anthocyaninup-regulation in ANRi lines were found (Fig. 4). This was mostclearly seen in the stigmata of the flowers. DMACA staining inthe ANRi lines is probably a result of the still active catechin bio-synthetic pathway, as LAR expression was not affected in theANRi lines (Fig. 1).





Histochemical analysis for flavonols in strawberry fruit wasperformed by application of DPBA and imaging under UV light.Strong and uniform up-regulation of flavonols in the entire fruitof ANRi lines in comparison with the untransformed control wasfound (Fig. 5). Vascular bundles were strongly colored yellow.

Metabolic profiling

Metabolite profiling of the ANRi lines (F22, F21, F17, F15 andF14) and the control was performed by LC-ESI-MSn analysis invarious tissues and at different fruit ripening stages. In transgenicstrawberry fruit, the anthocyanins pelargonidin and cyanidin glu-coside were already detected in fruits of the G1 stage, unlike thewild-type, where anthocyanins were first found in fruits of thewhite stage (Fig. 6). Cyanidin glucoside (300–1600 ppm) pre-vailed in immature fruits, whereas pelargonidin glucoside was thepredominant anthocyanin in ripe fruits of ANRi lines. It appearsthat the capacity for 3′-hydroxylation of the flavonoid skeleton islimited during strawberry fruit ripening, as the level of pelargoni-din glucoside (up to 12 000 ppm) exceeded the concentration ofcyanidin glucoside by a factor of 7.5 in the red ripening stage. Inthis stage, the levels of cyanidin glucoside in ANRi plants weresimilar 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 immaturefruit of ANRi line 17. However, in later stages, the concentrationleveled out and was indistinguishable from the levels in the othertransgenic lines. Flavonols (B-ring-dihydroxylated quercetin andmonohydroxylated kaempferol) behaved differently. Whereasquercetin glucuronide was strongly up-regulated in ANRi lines,kaempferol glucoside was down-regulated (Fig. S6).

The concentration of monomeric catechin was measured atvarious fruit ripening stages in untransformed control and ANRi

lines (Fig. 7). Catechin levels were reduced significantly in ANRifruits of the late ripening stages in comparison with wild-typefruits. By contrast, free epicatechin was not found in a quantifi-able concentration in any of the tissues analyzed.

Proanthocyanidins with a low DP can be analyzed directly byLC-ESI-MSn and distinguished by their mass spectra and reten-tion times. Here, four dimeric forms were found and quantified,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) cannotbe discriminated from those of LAR (afzelechin and catechin).The levels of dimeric proanthocyanidins were affected differentlyby the down-regulation of ANR (Fig. 8). Whereas (epi)catechin–(epi)catechin (isomer 2) and (epi)afzelechin–(epi)catechin (iso-mer 2) showed statistically significantly reduced levels in fruits ofANRi 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 wereobserved for (epi)afzelechin–(epi)catechin (isomer 1).

Analysis of higher proanthocyanidins by benzylthiol-mediated thiolysis

Proanthocyanidins can be analyzed for their starter and chain fla-van-3-ol subunits by thiolysis with benzylthiol. In addition, DPcan be calculated from the relative amounts of all subunits to thestarter unit (Table 1). Comparison of the subunits released bythiolysis 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 epiafzelechinchain subunits decreased from 59.2% and 3.6% to 23.1% and2.0%, respectively. By contrast, catechin starter and chain

Fig. 2 Gene expression analyses of flavonoid genes in tissues of the strawberry (Fragaria9 ananassa) ANRi line F17 relative to wild-type control andnormalized by ITS (internally transcribed spacer of ribosomal DNA) expression. The normalized expression levels are shown on a logarithmic scale tovisualize the down-regulation (values < 1). Error bars, � SD; significance 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,dihydroflavonol-4-reductase; FGT1, UDP-glucose:flavonoid-3-O-glucosyltransferase; FHT, flavanon-3-b-hydroxylase (syn. F3H); FLS, flavonol 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 thatANR products (epiafzelechin, epicatechin) are underrepresentedin the ANRi lines, whereas LAR products (catechin) are up-regu-lated. The modification of the starter and chain subunits alsoresulted in shorter proanthocyanidin chain lengths in the ANRilines, with a mean chain length of 3.8� 0.4 flavan-3-ol units perchain, in comparison with the untransformed control, with achain length of 5.8� 0.1 (Table 1).

Pollen viability

From petunia and maize mutants, it is known that flavonols areessential in these systems for pollen fertility. Flavonol-deficientmutants are self-sterile (Mo et al., 1992). The strongly increasedlevel of quercetin glucuronide in fruit tissue (data not shown) of

the ANRi lines suggested experimental testing of pollen viability,and some other flower traits (Table 2).

Pollen viability increased in ANRi lines (F14, F15, F21, F22)in comparison with the wild-type plant ‘Senga Sengana’ from42.7% to 72.5%, but other flower traits, such as the number ofpetals, sepals and anthers, were essentially unchanged, except forthe faint anthocyanin color of older petals.

Discussion

Transgenic lines

In the present study, 13 transgenic strawberry lines ofF.9 ananassa cv Senga Sengana were successfully establishedusing the transformation vector pBI-ANRi. The transgenic linesoriginate from independent transformation events and containbetween one and six T-DNA copies. Each line was successfully

Fig. 3 Anthocyanin phenotype of strawberry (Fragaria9 ananassa) ANRi line F17 in comparison with the untransformed control. Top left, flowerphenotype (left, untransformed control; right, ANRi line); top right, root phenotype (left, untransformed control; right, ANRi line); middle series, fruitphenotypes of untransformed control; bottom series, fruit phenotypes of ANRi line; G1–2, green fruit stages; W, white fruit stage; T, turning red stage;R, red stage. Bars, 1 cm.





propagated in vitro and transferred to the glasshouse. Glasshouseplants developed normally. No developmental differences wereobvious compared with the wild-type, except for red colorationof some transgenic plant tissues.

ANR down-regulation impacts on proanthocyanidinformation

The analysis of transformed lines for integration events and geneexpression analysis demonstrated that ANR was down-regulatedin a number of independent transformed strawberry lines(Fig. 2). The direct products of the down-regulated enzyme areepicatechin and epiafzelechin (Fig. 1). In histochemical analyseswith DMACA, their staining is overlain by catechin/afzelechinbiosynthesized by LAR, as the reagent cannot discriminate ste-reochemically different groups of flavan- 3-ols (Fig. 1). However,

the occurrence of polymers with both types of monomer stronglyargues 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 significantly reduced levels of mono-meric catechin in fruit of ANRi lines, whereas free epicatechincould not be found in strawberry fruit (Fig. 7). Subunit analysisof polymeric proanthocyanidins from ANRi lines revealed higherincorporation rates of catechin at the expense of epicatechinunits, probably owing to the reduced levels of epicatechin causedby ANR down-regulation (Table 1). The increased flow of cate-chin and/or its precursors into polymeric proanthocyanidins canexplain the small amounts of monomeric catechin in the trans-genic lines (Fig. 1). Similarly, levels of dimeric proanthocyaninscomposed of (epi)catechins were reduced in ANRi lines whencompared with controls (Fig. 8). However, the content of thestarter molecule epicatechin in polymeric anthocyanins wasalmost unaffected (Table 1). The proportion of epicatechin asstarter increased slightly from 1.2% in the control, by a factor of1.50, to 1.8% in ANRi lines. Likewise, the content of the startermolecule catechin rose from 16.0%, by a factor of 1.56, to25.0%. The total gain in starter molecules in comparison withchain (extension) units results from the reduced chain length ofpolymeric proanthocyanidins, which decreased from 5.8 units inthe control, by a factor of 1.52, to 3.8 units in ANRi lines. How-ever, major changes were observed for the composition of theextension units. Whereas, in wild-type plants, the epicatechinchain subunits accounted for 62.8%, this value decreased to25.1% in the ANRi lines. Thus, although the total production ofepicatechin 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 unchangedat 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 ectopicanthocyanin formation

The prominent effect on the other flavonoid classes, such asanthocyanins (ANS products) and flavonols (flavonol synthase(FLS) products) can mostly be explained by the artificial bottle-neck causing higher substrate concentration for these divergingbiosynthetic steps (Fig. 1). The observed effect of premature andectopic anthocyanin formation in strawberry fruit and stigmata,respectively (Fig. 3), also demonstrates that the enzymes requiredfor glycosylation of anthocyanidins are already present in earlystrawberry fruit development and stigmata, or that other gly-cosylating enzymes with lesser substrate specificity can take overwhen the ratio of competing enzyme activity changes by experi-mental down-regulation of ANR. A UDP-glucose:anthocyanidin3-O-glucosyltransferase (FaGT1) and two flavonol glucos-yltransferases (FaGT6 and 7) have been characterized fromF.9 ananassa (Almeida et al., 2007; Griesser et al., 2008a,b).They show distinct, but overlapping, enzymatic activities andspatiotemporal expression patterns. FaGT1, which adds glucosemoieties to pelargonidin and cyanidin, is weakly expressed in

Wild type

ANRi 17

No staining DMACA staining

Fig. 4 Anthocyanin phenotype (red stigmata) of flowers and receptacle aswell as histochemical analysis by dimethylaminocinnamicaldehyde(DMACA) staining for (epi)catechins–proanthocyanidins of strawberry(Fragaria9 ananassa) ANRi line F17 in comparison with untransformedcontrol. Top, flower and receptacle of untransformed control; bottom,flower and receptacle of ANRi line; left, visible (anthocyanin) phenotype;right, DMACA staining for catechins–proanthocyanins. Bar, 1 cm.

Fig. 5 Histochemical analyses for flavonols in strawberry(Fragaria9 ananassa) 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 fromFragaria. Thus, the enzyme involved in the accumulation ofquercetin glucuronide, which increased significantly in the ANRiline probably as a result of the higher availability of substrates,remains unknown.

Most prominent in the ANRi lines is the visible anthocyaninphenotype in all organs which form epicatechin/epiafzelechin inwild-type strawberries. These are mainly unripe fruits, stigmataof flowers and root tips (Hoffmann et al., 2012). As the down-regulation of ANRi led to the accumulation of anthocyanins inthese tissues, it appears that UDP-glucose:flavonoid-3-O-gluco-syltransferase (FGT1) and ANR compete for their common sub-strates pelargonidin and cyanidin. This is accompanied by thedepletion of leucoanthocyanidin substrate supply for ANS bystrong LAR activity, producing high levels of catechin and afzele-chin in unripe fruits. Similarly, the down-regulation of FaGT1 inF.9 ananassa caused both a reduction in anthocyanin pigmentsand accumulation of significant 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 flavonols. The concentration of the main flavonolquercetin glucuronide rose strongly according to histochemicalanalysis (Fig. 5) and analytical quantification by LC-MS analy-sis (Fig. S5). The minor flavonol kaempferol glucoside, whichreached only a 10-fold lower amount in strawberry fruits incomparison with quercetin glucuronide, showed reduced levelsin ANRi lines. Quercetin glucuronide is probably increased inANRi lines because of the accumulation of upstream flavonoidmetabolites. By contrast, lower levels of kaempferol gluco-side may be caused by depletion of the precursor UDP-glucoseby competing consumption by the induced anthocyaninformation (Fig. 1).

The observed higher pollen viability of ANRi lines argues for arole of flavonols in pollen fertility in strawberry, as described forpetunia and maize (Mo et al., 1992; Taylor & Jorgensen, 1992).Chalcone synthase-deficient petunia plants produced abnormalanthers devoid of flavonoid pigments. Although viable pollen wasproduced, pollen germination and tube growth were severelyreduced, indicating an effect of flavonoids on pollen fertility.

(a)

(b)

Fig. 6 Relative concentrations of cyanidinglucoside (a) and pelargonidin glucoside (b)in relation to an internal standard (4-methylumbelliferyl glucoside) in strawberry(Fragaria9 ananassa) fruits of different ANRilines (F22, F21, F17, F15 and F14) and thewild-type (wt). The mean values + SD werecalculated and plotted. Statistical significancewas calculated with the Wilcoxon–Mann–Whitney U-test and marked with asterisks(*, P ≤ 0.05; **, P ≤ 0.01). G1–2, green fruitstages (G1, green receptaculum tissuebetween achenes; G2, late green stage); W,white fruit stage; T, turning red stage; R, redstage.

Fig. 7 Relative concentrations of catechin in relation to an internal standard (4-methylumbelliferyl glucoside) in strawberry (Fragaria9 ananassa) fruits ofdifferent ANRi lines (F22, F21, F17, F15 and F14) and the wild-type (wt). The mean values + SD were calculated and plotted. Statistical significance wascalculated with the Wilcoxon–Mann–Whitney U-test and marked with asterisks (*, P ≤ 0.05; **, P ≤ 0.01). G1–2, green fruit stages; W, white fruit stage;T, turning red stage; R, red stage.





ANR down-regulation does not seem to affect fruit firmnessnegatively, 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 flavonoid biosynthesis (flavanon-3-b-hydroxylase (FHT, syn. F3H), FLS, ANS) (Halbwirth et al.,2006). Fruit firmness may be influenced by polyphenols whichare integrated into secondary cell walls: these may be flavan-3-ols,

but also hydroxycinnamic acids, diverging before the committedsteps of flavonoid biosynthesis (Braidot et al., 2008).

Statistically relevant changes have also been found at the geneexpression level for flavonoid genes, with increases or decreasesby factors of up to ten in various tissues. Such secondary effectson gene expression have also been observed by experimentaldown-regulation of other flavonoid genes in other systems, such

Fig. 8 Relative concentrations of dimericproanthocyanidins in relation to an internalstandard (4-methylumbelliferyl glucoside) instrawberry (Fragaria9 ananassa) fruits ofdifferent ANRi lines (F22, F21, F17, F15and F14) and the wild-type (wt). Themean values + SD were calculated andplotted. Statistical significance wascalculated with the Wilcoxon–Mann–Whitney U-test and marked with asterisks(*, P ≤ 0.05; **, P ≤ 0.01). G1–2, green fruitstages; W, white fruit stage; T, turning redstage; R, red stage.

Table 1 Concentration (%) of proanthocyanidin starter and chain subunits in three independent ANRi lines of strawberry (Fragaria9 ananassa) andwild-type control (mean values from four technical repetitions each) by benzylthiol thiolysis and subsequent analytical LC separation

ANR products LAR products

Chain length

StarterChain subunit

StarterChain subunit

Epicatechin3,4-trans-Epicatechinbenzylthioether

Epiafzelechinbenzylthioether Catechin

3,4-trans-Catechinbenzylthioether

3,4-cis-Catechinbenzylthioether

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� 0.1ANRi 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 flavan-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 (Malus9 domestica) (Szankow-ski et al., 2009) and flavonol glucosyltransferase in Arabidopsisthaliana (Yin et al., 2012). Interestingly, such gene expressionchanges are also observed by experimental manipulation offlavonoid levels with an enzyme inhibitor acting on 2-oxogluta-rate-dependent dioxygenases involved in flavonoid biosynthesis(Fischer et al., 2006). Taken together, this strongly argues for theexistence of feedback regulation mechanisms which regulate fla-vonoid gene expression via the sensing of flavonoid biosynthesisprecursors, 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 inFragaria9 ananassa. Epicatechin, the direct product of ANR,was consequently down-regulated as a chain extension unit in thepolymeric proanthocyanidins. However, as a starter unit of pro-anthocyanidins, the epicatechin proportion was found to remainunchanged, supporting the view of different epicatechin pools forstarter and chain subunits. An increasing concentration of the fla-vonol quercetin glucuronide was interpreted as a secondary effectvia the accumulation of intermediates and higher substrate avail-ability for this biosynthesis. Effects on gene expression of otherflavonoid genes were also observed and could be explained byfeedback regulation via flavonoid end products, intermediates orprecursors of flavonoid biosynthesis.

Acknowledgements

The Bayerisches Staatsministerium f€ur Umwelt und Gesundheitis gratefully acknowledged for supporting the Rosaceae rhizo-sphere project. Elizabeth Schroeder-Reiter (Ludwig-Maximilians-University Munich) is acknowledged for proof-reading of themanuscript as a native speaker. The authors acknowledgeI. Hiller, U. Hille, I. Polster and G. Klotzsche for technicalassistance.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Construction of vector pBI-ANRi.

Fig. S2 PCR-based detection of the nptII selectable marker genein 14 of 17 transgenic strawberry lines.

Fig. S3 PCR-based detection of the chimeric anr gene constructin 14 of 17 transgenic strawberry lines.

Fig. S4 Detection of integrated T-DNA copies in DNA of theANRi transgenic strawberry lines by Southern hybridization.

Fig. S5 Gene expression analyses of flavonoid genes in receptaclesof five independent strawberry ANRi lines (F22, F21, F17, F15and F14) relative to wild-type control.

Fig. S6 Relative concentration of quercetin glucuronide (top)and kaempferol glucoside (bottom) in fruits of different ANRilines (F22, F21, F17, F15 and F14) and the wild-type.

Table S1 Gene-specific primers: MYB, PAL, C4H, C4L, CHS,CHI, F3H, DFR, FLS, ANS, LAR, ANR, FGT1 and ITS (internaltranscribed spacer 26S-18S RNA, housekeeping transcript fornormalization)

Table S2 Tests for fruit firmness and ascorbic acid concentrationin ANRi lines of strawberry

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