Metabolic Interaction between Anthocyanin and Lignin ... · the South American domesticated...

Metabolic Interaction between Anthocyanin and LigninBiosynthesis Is Associated with Peroxidase FaPRX27in Strawberry Fruit1[W]

Ludwig Ring, Su-Ying Yeh, Stephanie Hücherig, Thomas Hoffmann, Rosario Blanco-Portales,Mathieu Fouche, Carmen Villatoro, Béatrice Denoyes, Amparo Monfort, José Luis Caballero,Juan Muñoz-Blanco, Jonathan Gershenson, and Wilfried Schwab*

Biotechnology of Natural Products, Technische Universität München, 85354 Freising, Germany (L.R., S.-Y.Y.,S.H., T.H., W.S.); Departamento de Bioquímica y Biología Molecular, Campus Universitario de Rabanales,Universidad de Córdoba, 14071 Cordoba, Spain (R.B.-P., J.L.C., J.M.-B.); Institut National de la RechercheAgronomique, Unité de Recherche 419, Unité de Recherche sur les Espèces Fruitères, Domaine de la GrandeFerrade, 33883 Villenave d’Ornon, France (M.F., B.D.); Investigación y Tecnología Agroalimentarias, Centre deRecerca en Agrigenòmica, Consejo Superior de Investigaciones Científicas-Investigación y TecnologíaAgroalimentarias-Universitat Autònoma de Barcelona-Universitat de Barcelona, 08193 Bellaterra, Cerdanyoladel Valles, Spain (C.V., A.M.); and Max Planck Institute for Chemical Ecology, 07745 Jena, Germany (J.G.)

Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of thephenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux throughthe pathway is not that well established. The aim of this study was to use a strawberry (Fragaria3 ananassa) microarray to investigategene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit.An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken toidentify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results werevalidated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberryfruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait.Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux fromanthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The listof the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used todevelop molecular markers to select phenolics-rich germplasm.

Anthocyanins, flavonoids (flavan-3-ols and flavonols),and phenylpropanoids are among the major phenolicsthat accumulate in ripe strawberry (Fragaria 3 ananassa)fruit (Fig. 1; Aaby et al., 2007; Fait et al., 2008; Tulipaniet al., 2008). Anthocyanins give rise to the red color ofstrawberry fruit, which attracts frugivores that help todisperse seeds (Willson and Whelan, 1990). Flavonols

are thought to function as protective chemicals againstUV-B light in fruit skin (Solovchenko and Schmitz-Eiberger, 2003), whereas proanthocyanidins such ascondensed (epi)catechin and afzelechin dimers (flavan-3-ols) contribute to defense and stress resistance (Panjehkehet al., 2010). The 1-O-acyl-Glc esters of cinnamic,4-coumaric, and caffeic acids (phenylpropanoids) mayserve as energy-rich substrates in plant secondarymetabolism (Lunkenbein et al., 2006a; Vogt, 2010), andellagic acid may play a role in protection from preda-tion and in plant growth regulation (Vattem and Shetty,2005).

In plants, phenolics arise from the shikimate, phe-nylpropanoid, flavonoid, anthocyanin, and lignin path-ways (Vogt, 2010). The shikimate pathway is requiredfor the biosynthesis of the aromatic amino acids (Fig. 1).In most plants, the biosynthesis of phenolics starts withphenylpropanoid formation (e.g. coumaric acid) fromthe primary metabolite Phe. The phenylpropanoidscan be further modified in many ways, including theconversion of monomeric phenylpropanoids to lignin

1 This work was supported by PLANT-KBBE FraGenomics, by theEuroinvestigacion Subprogram from PLANT_KBBE Call, providedby the Spanish Science and Technology Ministry (grant no.EUI2008–03770 to A.M.), by the Centre CONSOLIDER on Agrige-nomics and the Xarxa de Referencia en Biotecnología of the General-itat de Catalunya, and by the Deutsche Forschungsgemeinschaft(grant no. SCHW634/14 to S.-Y.Y. and W.S.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Wilfried Schwab ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.222778

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(insoluble phenolics) and the elongation and cycliza-tion of phenylpropanoids by the sequential addition ofthree molecules of malonyl-CoA to form the flavonoidsand anthocyanidins. Some of the genes and enzymesinvolved in phenolics biosynthesis were discovered andcharacterized in the model plants Arabidopsis (Arabi-dopsis thaliana), maize (Zea mays), and petunia (Petuniahybrida) and have been analyzed recently in Fragariaspp. (Lunkenbein et al., 2006a, 2006b; Almeida et al.,2007; Griesser et al., 2008a, 2008b; Schwab et al., 2011).The basic biosynthetic pathway leading to anthocyaninshas been almost completely elucidated (Ververidiset al., 2007). Considerable progress has also been madein understanding the regulation of this pathway, andtranscription factors that control the expression of thestructural genes have been characterized, but it is notyet known how this pathway is compartmentalized(Aharoni et al., 2001; Boudet, 2007; Allan et al., 2008;Gonzalez et al., 2008). Since phenolics are ubiquitous inall plant organs, they are an integral part of the humandiet and contribute to the nutritional quality of foods.

Identification of the genetic determinants governingboth fruit quality and agronomical traits is essential forthe sustained improvement of crops (Capocasa et al.,2008). The strawberry fruit is highly appreciated for itstasty flavor and nutritional value. However, fruit qualityattributes have been reduced or lost because breeding ofmodern cultivars has mainly focused on agronomicaltraits such as fruit size and yield. Thus, improvement ofstrawberry flavor, shelf life, and nutritional quality hasbecome an important factor in current breeding pro-grams, even when these quality attributes are controlledby a complex genetic background and are frequentlyassociated with negative agronomic characters (Zorrilla-Fontanesi et al., 2011). The challenge for breeders, who

want to produce berry fruit with high nutritional valuewhile maintaining an outstanding fruit quality, is notonly the knowledge of the single trait but also what isaffecting the variation and how different traits are corre-lated together. Since many different fruit traits have beensuccessfully modified with breeding strategies, breedingto increase one or more beneficial phytochemicals in fruitis likely to be achievable (Aharoni et al., 2004).

The 600 strawberry varieties found today stem fromfive or six original wild species and are members of theRosaceae family (Hancock, 1999). The common culti-vated strawberry has an octoploid genome (2n = 8x =56) that resulted from an accidental hybridization be-tween two related species, the scarlet strawberry (Fra-garia virginiana) that originated in North America andthe South American domesticated Fragaria chiloensis(Horvath et al., 2011). The origin of strawberry and theearly breeding practices reduced the initial geneticvariation. During the 200 years of strawberry breeding,the initial diversity increased due to the introgression ofwild strawberry germplasm or using unrelated pro-genitors, but these introgressions did not compensatefor the loss of diversity observed in modern strawberrycultivars (Gil-Ariza et al., 2009). Although genetic var-iation is low, the total phenolics content and composi-tion of phenolic compounds strongly differ betweenstrawberry genotypes (Rekika et al., 2005; Hernanzet al., 2007; Tulipani et al., 2008; Pincemail et al., 2012).Thus, cultivated strawberry varieties appear to be agood experimental model to elucidate new factorsregulating flux through the phenolics pathway, especiallybecause the genome sequence of Fragaria vesca (2n = 2x =14), one of the progenitors of strawberry, can be used as agenomic reference for the genus (Shulaev et al., 2011).Besides, the first microarray analysis of plant tissuewas successfully performed on RNA isolated fromfruits of the cultivated strawberry cv Elsanta, indicatingthat polyploidy is not a hindrance for this type ofanalysis (Aharoni et al., 2000).

In this study, an examination of the transcriptome,coupled with metabolite profiling analysis of differentstrawberry genotypes, was undertaken to reveal geneswhose expression levels correlated with altered phe-nolics composition. To exploit the impact of strawberrygenetic diversity on phenolics, 16 strawberry varietieswere used as a source of defined natural variation andas a resource for the identification of candidate regula-tory genes. The validity of the approach was strength-ened by the functional characterization of a peroxidasegene that showed the highest transcript level differencein phenolics-rich versus phenolics-poor genotypes. Inaddition, the role of the peroxidase gene in color andtotal polyphenols production was suggested by theidentification of quantitative trait loci (QTLs) linked tocolor in the region where the peroxidase gene is locatedand in two segregation populations. Analysis of trans-genic plants confirmed the metabolic interaction be-tween anthocyanin and lignin biosynthesis. Our resultsexpand the knowledge of mechanisms associated withlignin and phenolics biosynthesis and facilitate the

Figure 1. Scheme of the shikimate-phenylpropanoid-flavonoid-anthocyanidin-lignin pathway. Names (in boldface) and structures ofmetabolites quantified by LC-MS analysis are shown.

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development of novel strawberry cultivars with nutri-tionally superior properties.

RESULTS

Metabolite Profiling Analysis

The metabolite profiles of fully ripe fruits of 16 phe-notypically different strawberry varieties (Fig. 2) wereevaluated for their variation in phenolic compounds byliquid chromatography (LC)-mass spectrometry (MS;Fig. 1). The identity of the metabolites was confirmedby reference to authentic metabolites run under identical

conditions and to literature data (Fossen et al., 2004;Lunkenbein et al., 2006a, 2006b; Griesser et al., 2008a,2008b; Hanhineva et al., 2008). The major known phe-nolic metabolites were quantified in the positive andnegative MSmode by the internal standard method andwere expressed as per mil equivalent of dry weight,assuming a response factor of 1. This is a good methodof obtaining robust relative quantification rapidly (Fig.3). Metabolites, whose structures could not be unam-biguously elucidated, were not taken into account. Theheat map (Fig. 3A), which displays the relative levels of16 phenolic metabolites of the varieties, showed thateach variety accumulated a unique concentration pattern

Figure 2. Fruit phenotypes of strawberry varietiesthat were analyzed in the study and their identi-fier codes (ID). Some varieties are shown twice tovisualize the variations in size, shape, and colorand to show cross sections of the fruits.

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of metabolites (Fig. 2). The maximum and minimumlevels of these metabolites in the studied genotypes areshown in parentheses (Fig. 3A). Some varieties, such as49, accumulated only relatively low levels of varioussoluble phenolics in the fruits, whereas 3 containedrelatively high amounts of many metabolites, as can beseen by the number of blue and red squares, respec-tively. On the other hand, some metabolites, such aspelargonidin glucoside, occurred in high levels in nu-merous varieties, whereas its malonylated derivativewas only observed in a few genotypes, such as 42. Theconcentration values of the individual compounds wereused to calculate the cumulative levels of phenyl-propanoids, including ellagic acid, flavonoids, antho-cyanins, and total phenolics (Fig. 3B). Varieties 5 and 3were superior genotypes with regard to the total phe-nolics content due to their high amounts of anthocya-nins and phenylpropanoids. In contrast, genotypes 49and 21 produced only low levels of soluble phenolics.

Microarray Analysis

To couple the metabolite profiling analysis with anoverall examination of the transcriptome, we next

performed microarray analyses using selected straw-berry genotypes. In total, 17 unique comparative micro-array analyses were conducted on RNA isolated fromreceptacles of 12 different varieties that strongly differedin the level of one of 20 soluble phenolic metabolites,certain phenolic subgroups, and total phenolics, as de-termined by the metabolite profiling analysis (pairs ofgenotypes and the strategy of the analysis are detailed inFigure 3A and Supplemental Table S1). Oligonucleotide-based microarrays were manufactured from a Fastaarchive containing 18,152 uniESTs from a collection ofstrawberry sequences (Bombarely et al., 2010). The 17pairwise comparisons of transcript levels between geno-types with contrasting metabolites revealed, in total,26,416 differentially expressed ESTs (more than 4-fold,P , 0.01; Fig. 4A). Most of the mRNA expression levelsdiffered by less than 50-fold, but some transcriptsexceeded 200-fold and reached up to 925-fold expressionratios. The latter were thus selected as top candidategenes (Fig. 4B). For the most promising transcripts,protein-coding genes corresponding to the ESTs couldbe deduced from the genome sequence of F. vesca (TableI; Shulaev et al., 2011). The F. vesca database did notcontain protein-coding genes for five expressed mRNAs

Figure 3. Quantification of strawberryfruit phenolics and calculation of phe-nylpropanoid, flavonoid, anthocyanin,and total phenolics levels. Quantifica-tion of metabolites was performed byLC-MS analysis as per mil equivalents ofthe dry weight with the help of the in-ternal standard biochanin A (relativeconcentration). A, Heat map presenta-tion of the metabolite levels in differentvarieties. Varieties with the highest andlowest levels of individual metabolitesare shown in red and blue, respectively.Minimum and maximum levels of in-dividual metabolites are shown in pa-rentheses. B, Deduced levels (relativeconcentration as per mil equivalent ofthe internal standard) of phenylpropanoids,flavonoids, anthocyanins, and totalphenolics with SD of five biologicalreplicates.


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(FRA1441, FRA192B, FRA2718, FRA2456, and FRA3519).But FRA1441 clearly mapped on scaffold 0513150 (http://www.rosaceae.org/gb/gbrowse/fragaria_vesca_v1.0/),whereas the other sequences yielded ambiguous resultsand were not further considered. Besides the high dif-ferential expression levels of the candidates, they alsoappeared as differentially expressed (more than 4-fold)in most of the 17 comparative EST expression analyses,as indicated by the number of circles (Fig. 4B). Due toEST redundancy, gene 19544, which has been annotatedas a putative heme-containing PRX27 gene, showed upfive times among the most highly differentially expressedgenes, whereas gene 10776 and gene 19724 appeared twotimes. This confirms the good reproducibility of themicroarray platform. The role of gene 19544 was ana-lyzed in detail, as its expression might affect the qualityand quantity of phenolics.

FaPRX27 Is Expressed in Root and Red Fruit

Gene 19544 of the F. vesca genome encodes a putativeFaPRX27 peroxidase. Peroxidases catalyze the reduc-tion of hydrogen peroxide (H2O2) by taking electronsfrom various donor molecules. Class III plant peroxi-dases oxidize donors such as phenolics, lignin precur-sors, or secondary metabolites. The F. vesca genomeharbors 84 genes encoding putative heme-dependent

peroxidases (http://supfam.org/SUPERFAMILY/). Itis assumed that peroxidases are implicated in differentphysiological processes, such as lignification, suberiza-tion, cross linking of cell wall proteins, auxin catabolism,salt tolerance, defense against pathogen attack, andoxidative stress (Valério et al., 2004; Mathé et al., 2010;Lüthje et al., 2011). Therefore, it is important to de-termine the expression pattern of PRX27 to understandthe physiological roles and characteristics. FaPRX27gene expression was analyzed by quantitative PCR invegetative tissues, flowers, and fruits of different de-velopmental stages of cv Elsanta strawberry plants (Fig.5). Significant mRNA levels were only detected in rootsand red, ripe fruits, consistent with the results of themicroarray analyses. FaPRX27 transcript levels werenegligible in other tissues.

FaPRX27 Might Be Involved in Lignin Biosynthesis

The five ESTs contained on the microarray coveredmajor regions of gene 19544, but alignments with knownperoxidase sequences as well as BLAST searches in theNational Center for Biotechnology Information (NCBI)and EMBL yielded a different intron-exon structure, asproposed by the F. vesca genome database (Shulaev et al.,2011). Detailed analysis showed that gene 19544 pre-sumably consists of two different open reading frames

Figure 4. Selection of top candidate genes.A, Total ESTs that showed differential expression(more than 4-fold, P , 0.01) in 17 microarrayanalyses. Microarray analyses were performed ondifferent strawberry varieties that accumulatedopposite levels of phenolics, as determined bymetabolite profiling analysis (Fig. 3). B, ESTsshowing the highest levels of differential expres-sion. Protein-coding genes corresponding to theESTs were deduced from the genome sequenceof F. vesca (Shulaev et al., 2011). Gene 19544(boldface) appeared five times as highly differ-entially expressed due to EST redundancy.




http://www.rosaceae.org/gb/gbrowse/fragaria_vesca_v1.0/

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erroneously fused due to a misannotation of an intron.The intron removed a stop codon, eventually attachingthe peroxidase sequence to a second gene that codes fora Hyp-rich glycoprotein (Supplemental Figs. S1–S3). The39 and 59 untranslated regions contained in the ESTsconfirmed the assumption. Primers were designed thatamplified a 990-bp nucleotide sequence (FaPRX27) cor-responding to a 35.1-kD protein consisting of 329 aminoacids with a calculated pI of 8.47 (Fig. 6; SupplementalFig. S2). Eukaryotic linear motif search predicted a signalpeptide for the secretory pathway, spanning from aminoacids 1 to 26, and a transmembrane region from aminoacids 7 to 29 (http://elm.eu.org/search/; Dinkel et al.,2012). The full-length complementary DNA (cDNA) ofFaPRX27 was cloned by SmaI and XhoI substitution intothe pGEX vector and eventually introduced into Esche-richia coli Rosetta(DE3)pLysS. Cell extracts after induc-tion of the recombinant protein production contained anadditional 61-kD fusion protein (FaPRX27-GST [for glu-tathione S-transferase]), as shown by SDS-PAGE analysis(Supplemental Fig. S4). However, most induced proteinwas found as insoluble, suggesting that the GST-taggedFaPRX27 was mainly present in inclusion bodies. Theexpression of FaPRX27 without the GST tag was nottested.

Soluble protein fractions were used in enzyme assayswith ferulic acid, caffeic acid, and coniferyl alcohol inthe presence of H2O2, since the involvement of FaPRX27in lignin biosynthesis was assumed due to the observedinterference with the anthocyanin/flavonoid pathway.After addition of the substrates, a clear and rapid de-crease of absorption was observed, indicating the con-sumption of the phenylpropanoids (Fig. 7, A–C). Besides,the solutions turned yellow only when FaPRX27 wasadded. A guaiacol-containing solution quickly turnedpurple when FaPRX27was added but remained colorless

when an empty vector control extract was used (Fig.7D). The enzymatic activity was 0.02 mmol min21 mg21

total proteins with guaiacol as substrate (Fig. 7E). Ab-sorption did not change when cinnamic acid, 4-coumaricacid, vanillin, cinnamaldehyde, 4-hydroxybenzaldehyde,and 3,4-dihydroxybenzaldehyde served as substrates.It is assumed that in these cases, self-absorption of theprotein extract and similar absorption coefficients ofsubstrates and products interfere with the photometricdetermination of activity. Therefore, enzymatic assayswere additionally subjected to LC-MS analysis to detectpotential products (Supplemental Figs S5–S14). Thestarting material was almost completely consumed (lessthan 5% of the starting material remained unreacted)when ferulic acid and coniferyl alcohol were incubatedwith FaPRX27 (Fig. 8A; Supplemental Fig. S11). Caffeicacid, guaiacol, vanillin, and 3,4-dihydroxybenzaldehydewere also transformed, albeit the bulk of the substratesremained untouched (Supplemental Figs. S7, S9, S10,and S14). Although exact identification of the enzymaticproducts was not achieved, the interpretation of theirmass spectra allowed the conclusion that dimeric struc-tures were formed (Fig. 8). Compound 1 (386 g mol21;Fig. 8) appears to be a dehydrodimer of ferulic acid,whereas structures 2, 3, and 5 (342 g mol21) are probablyformed by decarboxylation of a dehydrodimer precursor(Ward et al., 2001). These data point to a function ofFaPRX27 in lignin biosynthesis, as dehydrodimers arethe major products of the initial steps of ferulic acidpolymerization by lignin peroxidase (Ward et al., 2001).Finally, total lignin content was determined in fruits ofgenotypes that accumulated extremely high and lowlevels of total phenolics (Fig. 9B). Varieties 3 and 5 con-tained significantly lower levels of the cell wall polymerthan 21 and 25 (Fig. 9A), consistent with the hypothesisthat FaPRX27 produced lignin at the expense of solublephenolic compounds. The results clearly support thehypothesis that FaPRX27 codes for a functional peroxi-dase putatively involved in the lignification in root andstrawberry fruit.

Table I. Candidate genes putatively correlated with phenolicsaccumulation in strawberry fruit

Gene No. Putative Annotation

Gene 19544 Peroxidase27 likeGene 23392 Cucumisin likeFRA1441 Maps on scf0513150FRA192B No homologyFRA2718 No homologyFRA2456 No homologyGene 21343 Expansin A8 likeGene 23054 Hothead likeGene 35152 Spidroin1 likeGene 10776 SRG1 likeGene 03515 Tasselseed2 likeFRA3519 No homologyGene 27098 GDSL esterase/lipase likeGene 19724 Heat shock protein likeGene 22502 Acyl-CoA-binding domain-containing proteinGene 33865 Ephrin A1 (LERK-1) likeGene 30399 Homeobox-Leu zipper ATHB15 likeGene 03472 Ubiquitin C-terminal hydrolase12 likeGene 00897 Defensin like

Figure 5. Relative FaPRX27 gene expression determined by quantita-tive PCR in fruit of different developmental stages, vegetative tissues,and flower of strawberry cv Elsanta. SG, Small green fruit; GW, green/white fruit; W, white fruit; T, turning fruit; R, red ripe fruit. An inter-spacer gene was used as an internal control for normalization, andwhite fruit was set at 1 as a reference (means 6 SE of five to six rep-licates with two sets of cDNAs). Relative changes are shown.


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A QTL Linked to Color Is Detected in the Regionof FaPRX27

To further investigate the potential role of the FaPRX27gene for the accumulation of phenolic compounds andagronomic traits such as color, we developed a QTL ap-proach using two segregating populations of the culti-vated strawberry. FaPRX27 is localized in the upperregion of the linkage group 3 (LG3) chromosome ofF. vesca (Shulaev et al., 2011). Gene information from thediploid genome was used on the cultivated strawberry

due to synteny and colinearity between diploid and oc-toploid Fragaria spp. genomes (Rousseau-Gueutin et al.,2008). Consequently, we considered the four linkagegroups, arbitrary named LG3a, LG3b, LG3c, and LG3d,holding the FaPRX27 gene. These four linkage groupsbelong to the homology group 3, which is homologousto LG3 of the diploid F. vesca. For mapping the gene inthe two segregating populations, we developed poly-morphic markers using simple sequence repeat (SSR)sequences localized close to the peroxidase gene. Thesemarkers were SSRCL317CGfB2 and SSR-PER for the

Figure 6. Protein sequence alignment of class III peroxidases. FaPRX27, Arabidopsis AtPRX27 (gi|15232058|ref|NP_186768.1),potato (Solanum tuberosum) SlTPX1 (gi|678547|gb|AAA65637.1), and horseradish (Armoracia rusticana) ArPrxC (gi|14603|emb|CAA00083.1) are shown. The alignment was performed by ClustalX with Geneious (http://www.geneious.com/web/geneious/).




http://www.geneious.com/web/geneious/

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Spanish F2 population Dover 3 Camarosa and theFrench F1 population Capitola 3 CF1116, respec-tively. The detected polymorphisms were mapped inpopulations, and markers were localized at the top ofone of the four homologous linkage groups of the

populations, LG3-D and LG3-A for the Spanish andFrench strawberry octoploid map, respectively (Rousseau-Gueutin et al., 2008).

QTLs were detected for all quantitative traits usingcomposite interval mapping for either Capitola or CF1116for a French strawberry population and Camarosa orDover for a Spanish population. Subsequently, we con-sidered only QTLs significantly superior to the log of theodds threshold (3.0 and 3.1 for the Spanish and Frenchsegregating populations, respectively) that were colo-calized with FaPRX27 (gene 19544) and therefore withthe SSR markers developed near the peroxidase gene(Table II). In the French segregating population, in theregion of the peroxidase gene, we identified QTLslinked to total polyphenol and total flavonoids on themale map. In the Spanish population, the peroxidasegene 19544 was colocalized with QTLs for epiafzelechin-pelargonidin glucoside and total flavonoids. In bothpopulations, in the region of the peroxidase gene, weidentified a QTL linked to visual fruit color decrease(Fig. 10).

Competition of the Anthocyanin/Flavonoid and LigninPathways for Common Precursors

Recently, we have shown that stably transformedantisense-silenced CHALCONE SYNTHASE (CHS) straw-berry plants produced significantly reduced levels ofanthocyanins in the fruits when compared with their wild-type counterparts but instead accumulated 4-coumaroyl-CoA-derivedmetabolites such as 4-coumaryl alcohol and4-coumaryl acetate (Lunkenbein et al., 2006b). Thesemetabolites are precursors of aroma chemicals such aschavicol, the reaction being catalyzed by EUGENOLSYNTHASE (Fig. 11D; Hoffmann et al., 2011). TransientRNA interference (RNAi)-mediated down-regulation ofCHS and simultaneous overexpression of EUGENOLSYNTHASE efficiently redirected the anthocyanin/flavonoid pathway to the phenolic volatiles (Hoffmannet al., 2006, 2011). To confirm the competition of theanthocyanin/flavonoid biosynthesis and lignin path-ways for common substrates, we down-regulated CHSexpression during strawberry fruit ripening by agro-infiltration of an inverted hairpin RNAi construct andanalyzed the effect on lignin content. One-half of a fruitwas agroinfiltrated, whereas the other half remaineduntreated (Fig. 11A). Silencing of CHS, as evidenced bythe loss of pigmentation, is accompanied by a signifi-cant increase in lignin content, which correlates withenhanced firmness of the fruits (Fig. 11B). Transcriptanalyses affirmed the reduced levels of CHS transcriptsdue to RNAi-mediated gene silencing (Hoffmann et al.,2006) and revealed the strong induction of FaPRX27expression upon agroinfiltration (Fig. 11C), whereasCINNAMOYL COENZYME A REDUCTASE (CCR) andCINNAMYL ALCOHOL DEHYDROGENASE (CAD)expression remained unaffected (data not shown). How-ever, fruits of stable CHS-deficient transgenic strawberrylines showed wild-type lignin levels and FaPRX27

Figure 7. Functional characterization of the FaPRX27 protein. A to C,Extracts from E. coli strain Rosetta(DE3)pLysS expressing FaPRX27were incubated with 140 mM H2O2 and 18 mM ferulic acid (A), caffeicacid (B), and coniferyl alcohol (C), while the absorbance was moni-tored at 310, 285, and 262 nm, respectively. Decreasing values in-dicate consumption of the substrates. E. coli extracts expressing anempty vector served as a control. D, The reaction mixture withFaPRX27 and the substrate guaiacol quickly turned purple, but itremained colorless when the empty vector control was added.E, FaPRX27 activity was determined photometrically by measuring theincrease in A470 due to oxidation products of guaiacol (see “Materialsand Methods”). F, Consumption of substrates by FaPRX27 was alsoquantified by LC-MS. Names and chemical structures of screenedsubstrates and percentage of converted substrate (in parentheses) after5 min of incubation are shown.


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expression levels (Lunkenbein et al., 2006b). RNAi-mediated down-regulation of FaPRX27 was unsuc-cessful, probably because the massive induction ofFaPRX27 transcription preceded RNAi. It can beconcluded that reduced flux through the anthocyanin/flavonoid pathway along with increased FaPRX27transcript levels significantly increased lignin contentand associated firmness at the expense of anthocyaninsin strawberry fruit (Fig. 11D).

DISCUSSION

Metabolite Profiling Analysis

As evidence accumulates about the positive healthbenefits of consuming a diet rich in phenolics, so doesour requirement for understanding the accumulation

of these compounds in staple foods. To correlate geneexpression profiles with phenolic compound accumu-lation in strawberry fruit, fruit phenolics were isolatedfrom diverse genotypes and major structurally identi-fied compounds were quantified by LC-MS (Fig. 3).Total phenolics content in the studied varieties showeda variation of a factor of less than 2, whereas the rangesof the levels of phenolic subgroups and in particularof individual compounds (shown in parentheses inFig. 3A) were much larger. For example, pelargonidinglucoside malonate was detected in one genotype in aconcentration of 10 mg g21 equivalent of the dry weightbut fell under the limit of detection (0.05 mg g21 equivalentdry weight) in most of the other varieties. Therefore, itseems that manipulation of the concentration of singlecompounds will be easier to achieve for breeders thanalterations in the total amount of certain groups of

Figure 8. LC-MS analysis of products formed byFaPRX27 from ferulic acid. A, The UV trace at280 nm shows the formation of novel compounds(1–7) by FaPRX27. B and C, E. coli extractsexpressing an empty vector (B) and assays with-out the addition of enzyme (C) served as controls.mAU, Milliabsorbance units. D, Mass spectra(-MS) and product ion spectra (-tandem MS of theindicated ion) of products 1 to 7 as shown in A.





secondary metabolites. There is probably more flexi-bility for the plant to divert biosynthetic pathways thanto increase the total biomass. Shifting biomass fromlignin biosynthesis to polysaccharide production hasbeen achieved in aspen (Populus tremuloides). Down-regulation of 4-coumarate-CoA ligase resulted in a45% decrease in lignin content and a concomitant 15%increase in cellulose content (Hu et al., 1999). Thesefigures were further increased to a 52% reduction inlignin content and a 30% increase in cellulose contentwhen coniferaldehyde 5-hydroxylase was also down-regulated (Li et al., 2003). Down-regulation of CCR intransgenic tobacco (Nicotiana tabacum) resulted in adecrease in lignin content and a concomitant increasein Xyl and Glc associated with the cell wall (Chabanneset al., 2001). Similarly, the redirection of different plantcarbon resources to the production of soluble phenolicsin fruit should be achievable (Griesser et al., 2008a).However, it is crucial that pathway manipulations donot interfere with fruit quality characteristics such astaste and firmness as well as defense against pathogensand insects.

Microarray Analysis

Combining metabolite profiling data with gene ex-pression analyses has provided unique and compre-hensive overviews of the transcriptional regulation ofmetabolic shifts, responses to plant hormones, as wellas adaption of stress tolerance mechanisms (Yonekura-Sakakibara et al., 2008; Janz et al., 2010; Kogel et al.,2010; Stushnoff et al., 2010; Rohrmann et al., 2011; Zifkin

et al., 2012). In this study, a custom-made oligonucleotide-based microarray platform (Roche-NimbleGen) wasemployed for the comprehensive investigation of dif-ferential gene expression in strawberry varieties thatwere selected by metabolite profiling analysis due totheir opposite levels of phenolics (Fig. 3; SupplementalTable S1). Seventeen unique comparative microarrayanalyses yielded 25 cDNAs that exceeded the 200-foldexpression ratio, representing 19 genes. The first micro-array analysis performed with cDNA from strawberryfruit dates from the year 2000 and led to the identifica-tion of a gene involved in fruit flavor formation (Aharoniet al., 2000). Later, differential gene expression by micro-array was determined in strawberry achene versus re-ceptacle tissue and in fruit compromised by oxidativestress (Aharoni and O’Connell, 2002; Aharoni et al.,2002). In these studies, similar high changes in expres-sion levels (up to 300-fold) were observed for genes indifferent tissues of the same genotype. Detailed inspection

Figure 9. A, Lignin content of strawberry geno-types with high levels (black bars) and low levels(white bars) of soluble phenolics. B, Content oftotal soluble phenolics. C, Relative expressionlevels of FaPRX27 in different genotypes calcu-lated from transcript levels determined by micro-array analysis. FaPRX27 levels in genotypes 3 and5 did not differ and were set as 1. Transcript levelsin 21 and 25 were calculated frommicroarray dataobtained from the comparisons of genotypes 5/21,3/25, 5/25, and 21/25. D, Simplified scheme toshow the proposed effect of FaPRX27 expressionon the total amount of soluble phenolics.

Table II. MapQTL summary for color 2009 in LG3-D of the Spanishsegregating population

Map LOD Percentage Expl Add Dom Locus

0 5.5 13.4 20.27 0.22 CL317CGfB27.8 0.2 7.3 20.16 20.08 g676CGdD1

26.7 3.8 20.8 20.11 20.32 g235eD36.7 3.9 19.3 20.01 20.39 BFACT43A3+53.3 1.9 11.6 20.28 0.14 CFACT74B259.8 1.9 12.7 20.49 0.01 CFACT37CD3+74.1 18.0 75.5 20.46 0.54 CFACT42B

Add, Additive effects; Dom, dominance effects; Expl, explained.


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of the most highly differentially expressed ESTs in ourstudy and alignment with the F. vesca genome revealedredundancy of the ESTs represented on the array butconfirmed the reliability of the analysis. The top can-didates (Supplemental Fig. S15) appeared in a numberof comparative analyses as highly differentially expressedgenes, which demonstrates their importance in phenolicsmetabolism. Gene 19544, annotated as putative FaPRX27,was remarkable, as it showed up five times among thetop candidates and displayed the highest difference inexpression level, but it has not been described in previousstudies. Thus, FaPRX27 was investigated further, sinceperoxidases are assumed to function in lignin biosynthesis,

which is connected with the pathway of soluble phenolicsvia coumaroyl-CoA (Vassão et al., 2008).

FaPRX27

Peroxidases (EC 1.11.1.X) are oxidoreductases thatcatalyze the oxidation of various substrates by reducingH2O2 to water. Phenolic compounds (e.g. lignin pre-cursors and secondary metabolites) can serve as electrondonor molecules. The subcategory of heme peroxidasescarry a protoporphyrin IX complexed with Fe(III) (theferric form in the ground state) in their active sites andshare a very similar three-dimensional structure (Mathéet al., 2010). Recently, a separate, additional hydroxyliccycle of peroxidases, which leads to the formation ofvarious reactive oxygen species, has been proposed(Cosio and Dunand, 2009).

Land plants contain a large number of class IIIperoxidases (EC 1.11.1.7). The Arabidopsis, rice (Oryzasativa), and F. vesca genomes harbor 73, 138, and 84genes encoding peroxidases, respectively (Passardiet al., 2004; Valério et al., 2004; http://supfam.org/SUPERFAMILY/). The large number of PRX genes inrice resulted from various duplication events that weretentatively traced back (Passardi et al., 2004). Com-parison with the predicted number of heme-containingperoxidase genes in sequenced plant genomes showedthat F. vesca and Arabidopsis contain the lowest numberof PRX genes (http://supfam.org/SUPERFAMILY/).As a consequence of the large number of genes and thetwo possible catalytic cycles, it is assumed that class IIIplant peroxidases are involved in a broad range ofphysiological processes like cell elongation, cell walldifferentiation, or defense. They function in cell wallmetabolism (Marjamaa et al., 2009), wound healing,auxin catabolism, removal of H2O2, oxidation of toxicreductants, defense against pathogen and insect attack,as well as symbiosis and normal cell growth (Cosioand Dunand, 2009; Lüthje et al., 2011). Their roles inthe modification of cell wall structures may includesuberin polymerization, cross linking of the structuralnonenzymatic proteins such as expansins, catalyz-ing the formation of diferulic acid linkages betweenpolysaccharide-bound lignin or ferulic acid residuesin polysaccharides, and the production of hydroxylradicals with the ability to cleave cell wall polymers(Marjamaa et al., 2009). Few peroxidase genes exhibitan organ-dependent expression, while others are ac-tive in the whole plant or expressed in lignifying tissue(Valério et al., 2004). FaPRX27 transcripts were exclu-sively found in root and fruit, consistent with theexpression pattern of its closest homolog from theArabidopsis genome, AtPRX27 (Valério et al., 2004;Cosio and Dunand, 2009).

Lignification is one of the functions classically at-tributed to class III peroxidases (Quiroga et al., 2000;Marjamaa et al., 2009). It is a cell wall-fortifying pro-cess that occurs in xylem tissue in a scheduled mannerduring tissue differentiation. A number of studies have

Figure 10. QTL analyses of a Spanish and a French segregating pop-ulation of strawberry. A, Linkage group 3 (LG3-D) of a Spanish pop-ulation map and color QTL detection. B, Male LG3-A of a Frenchpopulation and QTL linked to color, total flavonoids, and total poly-phenols. Markers developed to localize FaPRX27 are boxed. cM, Cen-timorgan; LOD, log of the odds.









implicated different peroxidase genes from Arabi-dopsis (AtPRX13,AtPRX17,AtPRX30,AtPRX42,AtPRX53,AtPRX55, AtPRX64, AtPRX66, and AtPRX71) in thelignification of specific tissues, but AtPRX27 is notamong them (Cosio and Dunand, 2009). Besides, theparticipation of class III plant PRXs in lignin formationhas been studied in many other plant species and cellculture systems, most importantly tobacco, Zinnia ele-gans, tomato (Solanum lycopersicum), and both gymno-sperm and angiosperm tree species (Marjamaa et al.,2009). Most of these studies are based on finding PRXenzymes with suitable catalyzing properties and locali-zation of proteins or gene expression in lignifying xylem.However, there are only a few examples of transgenicplants or mutant lines with modified PRX expressionand, consequently, altered lignification patterns probablydue to the large number and possible functional redun-dancy of PRX enzymes (Mansouri et al., 1999).

FaPRX27 represents a classical class III plant per-oxidase (Veitch, 2004). The basic protein (calculated pIof 8.47) harbors a predicted signal peptide for the se-cretory pathway (amino acids 1–26), a predicted trans-membrane region (amino acids 7–29), six predictedN-glycosylation sites (amino acids 82–87, 95–100, 151–156,168–173, 209–214, and 216–221), and assumed phos-phorylation sites (http://elm.eu.org/search/; Dinkelet al., 2012). Most of the putative membrane-boundPRXs were predicted as secretory proteins (Lüthjeet al., 2011). Besides cleavable N-terminal signal pep-tides, transmembrane domains have been calculated byseveral prediction programs for a number of class IIIperoxidases. Glycans could stabilize the proteins andaffect substrate access in peroxidases because of theirlarge size (Mathé et al., 2010). Key features of the centralactive site that have been identified in the horseradishperoxidase (ArPrxC) three-dimensional structure are

Figure 11. Competition of the anthocyanin andlignin pathways for common substrates in fruits ofcv Elsanta. A, Phenotypes of a fruit whose left halfremained untreated (a; red) whereas the right halfwas injected with A. tumefaciens containing anFaCHS-inverted hairpin RNA construct (b; white)and of an untreated wild-type control fruit (WT; c)14 d after injection. B, Lignin content and fruitfirmness were measured 14 d after infiltration.The box plots show data from different groupswith the following biological replicates: firmnessof red (n = 8), white (n = 8), and wild-type (n =10) fruit and lignin content of each group (n = 6).N, Newton. C, Relative expression of FaPRX27 inresponse to agroinfiltration. Fruits were infiltratedwith A. tumefaciens cells in the turning ripeningstage and harvested 1, 4, 8, and 10 d after in-jection. Wild-type (untreated) fruits were used ascontrols. Expression levels in control (gray col-umns) and agroinfiltrated fruits (black columns)were monitored by quantitative real time-PCRusing specific primers for FaPRX27 and theinterspacer gene. The latter was used as an in-ternal control for normalization. The control fruit(1 d) was used as the reference (set to 1). Valuesare means 6 SE of six replicates from two inde-pendent fruits and are shown as relative changes.D, Phenolics pathway showing the relationshipof flavonoids/anthocyanins, lignin, and chavicolformation in strawberry fruit.


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http://elm.eu.org/search/


completely conserved in the amino acid sequence ofFaPRX27, namely Arg-65, Phe-68, His-69, Asn-96, His-194, and Pro-164, that accepts a hydrogen bond fromreducing substrates and determines peroxidase sub-strate specificity (Fig. 5; Veitch, 2004). The proximal(Thr-195 and Asp-247) and distal (Asp-70, Gly-75, Asp-77,and Ser-79) Ca2+-binding sites are also maintained.Residues 70 to 97 are important for activity in plantperoxidases, as Asn-96 in this loop is hydrogenbonded to the active-site distal His-69, thereby ori-enting the hydrogen-bonding network in the distalcavity and regulating the acid dissociation constant ofthis amino acid. A conserved Glu-93 participates in thesame hydrogen-bonding network, which also involvesthe distal Ca2+ (Cosio and Dunand, 2009). Among thenine Cys residues found in FaPRX27, eight occur inconserved positions consistent with four potential di-sulfide bridges (Mathé et al., 2010).Although there are large variations of intron number

and length among PRX class III paralogs and ortho-logs, intron positions and phases are remarkably con-served (Mathé et al., 2010). This suggests that intronloss, or marginally intron gain, probably occurred duringthe expansion of the gene family in some plant lineages.The coding sequence of FaPRX27 is disrupted by threeintrons at perfectly conserved positions according tothe classical pattern of peroxidase genes (SupplementalFig. S3).Enzymatic assays confirmed the role of FaPRX27 in

lignin biosynthesis, as precursors such as coniferylalcohol, ferulic acid, and caffeic acid were transformedinto dehydrodimers in the presence of H2O2. Ferulicacid dehydrodimers produced in vitro and in vivo byplant PRXs have been extensively characterized (Wardet al., 2001). Different regioisomers resulting from rad-ical coupling are found in plant cell walls, where theycross link polymers, providing strength and rigidity.The units resulting from the monolignols 4-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol, whenincorporated into the lignin polymer, are called H, G,and S units, respectively (Vanholme et al., 2010). Ligninextracted from strawberry fruit is mainly composed ofG (65%–80%), S (8%–21%), and traces of H (1%–3%)units, as can be expected for angiosperm dicot lignin(Vanholme et al., 2010) and was confirmed by ligninextracted from fruits (Bunzel et al., 2005). Thus, the highproportion of guaiacol (G) groups in strawberry fruitlignin correlates well with the substrate specificity ofFaPRX27.Lignin formation in fruit may contribute to their

firmness, as microarray analyses have shown thatgenes involved in the degradation of pectin and cel-lulose, two structure-defining and stabilizing polymercomponents of fruits, were not differentially expressedin three strawberry varieties producing fruits with firm(cv Holiday), medium (cv Elsanta), and soft (cv Gorella)structures. Interestingly, two genes encoding proteinscatalyzing successive reactions in lignin metabolism(CCR and CAD) showed the highest differences in ex-pression levels (Salentijn et al., 2003). These results were

confirmed by a second group in other varieties (Carboneet al., 2006). Similarly, during postharvest ripening ofloquat (Eriobotrya japonica) fruit, firmness increased andshowed a positive correlation with the accumulation oflignin in the flesh and increased activities of CAD andPRX (Cai et al., 2006).

Furthermore, QTL analysis showed that FaPRX27 islinked to a region implicated in fruit color decrease. Thisexperimental approach is functionally relevant andconfirms FaPRX27 as a functional lignin peroxidaseputatively involved in the polymerization of solublephenolics in planta. The gene product competes withenzymes of the flavonoid and anthocyanin pathwayfor substrates (Fig. 9D) and thus determines the in-tensity of the red fruit color. Coumaroyl-CoA repre-sents a common intermediate, as it can be converted toPRX substrates (ferulic acid and coniferyl alcohol) andserves as a precursor of flavonoids and anthocyanins.Our study showed that varieties expressing FaPRX27 tohigh levels accumulated low amounts of soluble phe-nolics but contained significantly higher concentrationsof lignin than genotypes showing extremely low tran-script levels. Similarly, overexpression of a basic per-oxidase (SlTPX1) in tomato yielded a 40% to 220%increase of lignin content in the leaf in transgenic plants(Mansouri et al., 1999). In contrast, in Arabidopsis,silencing of hydroxycinnamoyl-CoA shikimate/quinatehydroxycinnamoyl transferase resulted in the repres-sion of lignin synthesis and led to the redirection of themetabolic flux into flavonoids through chalcone syn-thase activity (Besseau et al., 2007). Hypolignified stemsof Arabidopsis resulting from the concurrent down-regulation of CADc, CADd, and CCR1 accumulatedhigher levels of flavonol glycosides, which suggests aredirection of the phenolic pathway (Thévenin et al.,2011). The maize R2R3-MYB factor ZmMYB31 re-pressed lignin biosynthesis in transgenic Arabidopsisand redirected the phenylpropanoid carbon flux towardthe biosynthesis of anthocyanins (Fornalé et al., 2010).Finally, we showed by RNAi-mediated down-regulationof CHS, which is accompanied by a significant increase ofFaPRX27 transcript levels (more than 1,500-fold; Fig. 11C)due to agroinfiltration, that the accumulation of antho-cyanins is considerably decreased (Fig. 11A) at the ex-pense of lignin production (Fig. 11B). Overall, the resultsdemonstrated that manipulation of the lignin biosynthesispathway enzymes can yield strawberry fruit with in-creased levels of soluble phenolic compounds and thusprovide potential health benefits.

CONCLUSION

Comparison of the transcript patterns of differentstrawberry genotypes combined with metabolite pro-filing analysis revealed novel candidate genes thatmight affect the accumulation of flavonoid and an-thocyanin in strawberry fruit. Interestingly, neitherstructural genes nor transcription factors of the phe-nolics pathway appeared among the top 20 candidates.







The majority of the novel genes have not been impli-cated in the secondary metabolism. A putative perox-idase gene was analyzed in detail to test the validity ofthe approach. FaPRX27 encodes a functional enzymethat is required for the polymerization of phenyl-propanoids in ripening strawberry fruit and thus com-peting with flavonoid/anthocyanin pathway enzymesfor common precursors. In this respect, FaPRX27 couldbe a trigger in balancing the flux toward soluble andinsoluble (lignin) phenolic compounds. Future studieswill show whether other candidates affect phenolicsaccumulation in a similar or different fashion.

MATERIALS AND METHODS

Chemicals

Except where otherwise stated, all chemicals, solvents, and reference com-poundswere obtained from Sigma-Aldrich, Fluka, Riedel deHaёn,Merck, or Roth.

Sample Extraction

Ripe strawberry (Fragaria 3 ananassa) fruits were harvested in 2009 from 16selected cultivars (Fig. 2) cultivated in the south of Spain and in 2010 from twosegregating populations, an F2 and a “pseudo test-cross F1,” from Spain andFrance, respectively. Fruits were individually frozen, lyophilized for 48 h, andhomogenized with a mill (Retsch MM 200) to a fine powder. An aliquot of50 mg of lyophilized fruit powder was used for each of the three biologicalreplicates. Biochanin A (250 mL of a solution in methanol, 0.2 mg mL21) wasadded as an internal standard, yielding 50 mg of internal standard in eachsample. After the addition of 250 mL of methanol, vortexing, and sonicationfor 10 min, the sample was centrifuged at 16,000g for 10 min. The supernatantwas removed, and the residue was reextracted with 500 mL of methanol. Thesupernatants were combined, concentrated to dryness in a vacuum concen-trator, and redissolved in 35 mL of water. After 1 min of vortexing, 10 min ofsonication, and 10 min of centrifugation at 16,000g, the clear supernatant wasused for LC-MS analysis. Each extract was injected twice (technical replicate).

LC-Electrospray Ionization-Multiple Stage MassSpectrometry Analysis

Samples were analyzed on an Agilent 1100 HPLC/UV system (AgilentTechnologies) equipped with a reverse-phase column [Luna 3u C18(2) 100A,150 3 2 mm; Phenomenex] and connected to a Bruker esquire3000plus ion-trapmass spectrometer (Bruker Daltonics). As mobile phase, 0.1% formic acid inwater (A) and 0.1% formic acid in methanol (B) were used. Injection volumewas 5 mL, and flow rate was 0.2 mL min21. A gradient was applied thatstarted at 0% B and went to 50% B in 30 min. Within the next 5 min, B wasincreased to 100%, where it was kept for 15 min. Afterward, B was decreasedto 0% within 5 min. These initial conditions were kept for 10 min for systemequilibration. UV signals were detected at 280 nm. MS spectra were recordedin alternating polarity mode. Nitrogen was used as nebulizer gas at 30 p.s.i.and as dry gas at 330°C and 9 L min21. The electrospray ionization voltage ofthe capillary was set to 24,000 V, the end plate to 2500 V, the skimmer to40 V, and the capillary exit to 121 V. The full scan ranged from 100 to 800mass-to-charge ratio with a resolution of 13,000 mass-to-charge ratio persecond. Ions were accumulated until an ion charge control target of 20,000(positive mode) or 10,000 (negative mode) was achieved or the maximum timeof 200 ms was reached. For tandem MS, helium was used as the collision gasat 4 3 1026 mbar and a collision voltage of 1 V. Data were analyzed with DataAnalysis 5.1 software (Bruker Daltonics). Metabolites were identified bycomparing their retention times and mass spectra (MS and tandem MS) withthose of measured reference compounds. Phenylpropanoyl Glc esters wereenzymatically synthesized with FaGT2 (Lunkenbein et al., 2006a). Pelargo-nidin 3-O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-glucoside,kaempferol 3-O-glucuronide, kaempferol 3-O-glucoside, catechin, and epi-catechin were obtained from Roth. Proanthocyanidins and pelargonidin 3-O-glucoside-6-O-malonate were isolated from strawberry and identified

according to Fossen et al. (2004). Statistical evaluation was performed usingindependent Student’s t test of SOFA Statistics (Paton-Simpson & Associates).The major known phenolic metabolites were quantified in the positive (an-thocyanins) or negative (phenylpropanoids and flavonoids) MS mode by theinternal standard method and were expressed as per mil equivalent of dryweight assuming a response factor of 1. The metabolite concentrations did notalways lie within the linear range of the detector, and the calculation of theirrelative levels did not allow immediate comparison with absolute levels forphenolics provided by other studies, but the method offered the advantage ofobtaining relative values in a short period of time that is sufficient to rank thevarieties according to their metabolite levels (Fig. 3). The consumption of sub-strates by FaPRX27 was also quantified by LC-MS under the same conditions.Samples were analyzed prior to the addition of FaPRX27 and 5 min after theaddition of FaPRX27. Ion counts of the substrates were used for the calculation.

Quantitative PCR Analysis

Two grams of frozen plant material was ground to a fine powder in liquidnitrogen using a mortar and pestle for total RNA extraction. Total RNA wasprepared as described (Liao et al., 2004). First-strand cDNA was synthesizedfrom 1 mg of DNase I (Fermentas)-treated total RNA by Moloney murineleukemia virus reverse transcriptase H2 (Promega) and random hexamerprimer according to the manufacturer’s instructions. Real-time PCR wasperformed on a 96-well reaction plate (Applied Biosystems) with theStepOnePlus real-time PCR system (Applied Biosystems) using Fast SYBRGreen Master Mix (Applied Biosystems) to monitor double-strandedcDNA synthesis. Two microliters of a 50-fold dilution and 2 mL of a4,000-fold dilution cDNA were used as templates for targeting FaPRX27and interspacer genes, respectively. The following primers were used:FaPRX27 gene-specific primers (forward, 59-ATTTCCATGATTGCTTT-GTCA-39; reverse, 59-CAACGGCTAAGATGTCAGAAC-39) and inter-spacer primers (forward, 59-ACCGTTGATTCGCACAATTGGTCATCG-39;reverse, 59-TACTGCGGGTCGGCAATCGGACG-39). PCR (three technicalreplicates) was performed using the following thermal cycling conditions:95°C for 10 min (holding stage), then 95°C for 15 s and 60°C for 1 min (40 cycles),followed by 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s (melt curve stage).Relative gene expression was quantified using the 2-DDCT method (Livak andSchmittgen, 2001), and values were calculated as fold change of each samplerelative to the selected reference sample (white fruit).

Construction of the FaPRX27 Expression Plasmid

The full-length sequence of FaPRX27 was isolated from cDNA of red fruitof strawberry (cv Elsanta) using primers for_19455 (59-ATGGCTGCTACTT-CAA-39) and rev_19455 (59-CTAATTGATCTTGCTGC-39). The amplificationproduct was A tailed and cloned into the pGEM-T Easy (Promega) vector, andthe ligation product was transformed into Escherichia coli NEB10 beta (NewEngland Biolabs). The identity of the cloned gene was confirmed by se-quencing the complete insert (MWG Biotech) and by restriction enzyme di-gestion with PstI/SacI and NotI. SmaI and XhoI restriction sites wereintroduced by sense and antisense primers, respectively, and the PCR productwas cloned in frame with a coding region for a C-terminal GST tag into pGEXexpression vector (GE Healthcare). Correct insertion was confirmed by se-quencing, and FaPRX27 expression vector was finally transformed into E. coliRosetta(DE3)pLysS (Merck).

Heterologous Protein Expression

E. coli Rosetta(DE3)pLysS cells carrying the FaPRX27 expression vector weregrown overnight at 37°C in Luria-Bertani medium containing 50 mg mL21 car-benicillin and 34 mg mL21 chloramphenicol. The next day, the cultures werediluted to an optical density at 600 nm of 0.06 with Luria-Bertani mediumcontaining the appropriate antibiotics in a final volume of 400 mL. This culturewas grown at 37°C to an optical density at 600 nm of 0.3 to 0.4, and 200 mL of 1 M

isopropylthio-b-galactoside was added to induce protein expression. Afterovernight (19–24 h) incubation at 16°C to 18°C, cells were harvested by cen-trifugation and stored at 280°C. The pellet was resuspended in 10 mL ofextraction buffer (100 mM potassium phosphate buffer, pH 6.6, 0.5% TritonX-100, and 2 mM EDTA) containing 10 mL of the proteinase inhibitor phenyl-methylsulfonyl fluoride (10 mM in 2-propanol). Cells were lysed by sonication(3 3 30 min, 18% intensity; Bandelin Sonoplus) followed by vigorous vortexing.


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Crude protein extract was centrifuged (6,000g at 4°C), and the supernatant fi-nally was used in PRX enzyme assays.

Enzyme Assays

Enzyme activity was assayed according to a published procedure (Vitaliet al., 1998) in solutions consisting of 2.8 mL of buffer (0.1 M potassiumphosphate buffer, pH 7.0), 50 mL of substrate (18 mM), 50 mL of H2O2 (30%,1:100 diluted, optical density at 240 nm = 0.4), and 100 mL of crude FaPRX27extract (1 mg mL21). Reactions were monitored photometrically at thewavelength of maximum absorption of the respective substrates. In addition,after 5 min, 50 mL of the assays was withdrawn, and 25 mL of methanol wasadded, vortexed, and centrifuged (14,000g for 5 min). The supernatant wasanalyzed by LC-multiple stage mass spectrometry. As a control, Rosetta(DE3)pLysS cells were transformed with an empty pGEX vector, and the resultingprotein extract was assayed under the same conditions. As an additionalcontrol, assays were conducted without the addition of protein extracts.FaPRX27 activity was quantified photometrically by measuring the increase inA470 due to the formation of oxidation products of guaiacol. The 300-mL re-action mixture contained 230 mL of 0.1 M potassium phosphate buffer (pH 7.0),25 mL of 18 mM guaiacol, 25 mL of 9.8 mM H2O2 (30% solution), and 20 mL ofcrude protein extract. The reaction was started by the addition of the proteinextract at room temperature. Enzymatic activity was calculated with an ex-tinction coefficient of 26.6 mM

21 cm21 at 470 nm (Vitali et al., 1998). Values aremeans 6 SE of three replicates.

Quantification of Lignin

Extraction of lignin was adapted from published procedures (Meyer et al.,1998; Franke et al., 2002). Fifty milligrams of lyophilized fruit powder wassuspended in 1.5 mL of 0.1 M phosphate buffer (pH 7.2), sonicated for 1 min,and kept agitated at 500 rpm and 40°C for 30 min. The suspension wascentrifuged for 30 min at 16,000g, and the supernatant was discarded. Thesample was extracted a second time with 1.5 mL of 0.1 M phosphate buffer (pH7.2). The residue was washed twice with 1.5 mL of 80% ethanol, sonicated for1 min, and incubated at 80°C for 10 min at 500 rpm. The sample was centri-fuged at 16,000g for 10 min, and the supernatant was removed. The residuewas then washed with 1.5 mL of acetone, sonicated for 1 min, and centrifugedfor 30 min at 16,000g, and the supernatant was discarded. The residue wasincubated with 950 mL of 1 M sodium hydroxide solution for 16 h at roomtemperature. The solution was neutralized with 950 mL of 1 M hydrochloricacid and centrifuged at 16,000g for 10 min. The residue was washed twice with1.5 mL of water followed by sonication for 1 min and centrifugation for 15 minat 16,000g. Quantification of lignin was done according to a published report(Campbell and Ellis, 1992). The pellet was suspended with 750 mL of water,250 mL of concentrated hydrochloric acid (32%), and 100 mL of thioglycolicacid and incubated for 3 h at 80°C. After centrifugation (5 min at 16,000g),1 mL of water was added to the residue, sonicated for 1 min, and centrifuged.The residue was dissolved in 1 mL of 1 M sodium hydroxide solution for 12 hof agitation at 500 rpm at room temperature. After centrifugation (5 min at16,000g), the supernatant was placed into a new tube, mixed with 200 mL ofconcentrated hydrochloric acid (32%), and kept for 4 h at 4°C for precipitationof lignin. After centrifugation (10 min at 16,000g and 4°C), the residue wasfinally dissolved in 1 mL of 1 M sodium hydroxide solution and its absorbancemeasured photometrically at 280 nm, being diluted if necessary. A calibrationcurve was prepared with 1, 2.5, 5, 7.5, and 10 mg of hydrolytic lignin followingthe same procedure (Sigma-Aldrich). Six biological replicates were analyzed.

RNAi-Mediated Gene Silencing

For transient transfection of strawberry fruit, Agrobacterium tumefaciensstrain AGL0 suspensions containing pBI-CHSi (Hoffmann et al., 2006) wereinjected into one-half of several fruits, whereas the other one-half remaineduntreated. The octoploid strawberry cv Elsanta was used for transfections.Fruits remained attached to the plants after agroinfiltration. A detailed de-scription of the agroinfiltration procedure (timing and sampling) has beenpublished (Hoffmann et al., 2006).

Determination of Fruit Firmness

Fruit firmness was determined by a TA-XT2i texture analyzer (Singh andReddy, 2006). The measuring force was made with a probe of 0.5 mm in

diameter to penetrate the surface of the fruit. Each fruit was penetrated at aspeed rate of 1 to 10 mm s21. Based on the bioyield point, the maximum forcedeveloped during the measurement was recorded and expressed in Newton.Each sample was measured twice on the two opposite sides.

Bioinformatics of EST Sequences

A total of 29,741 raw strawberry EST sequences were processed with theprograms included in the EGassemblerWeb page (http://egassembler.hgc.jp/;Masoudi-Nejad et al., 2006). This included a masking process in which poly(T)tails, vector and adapter sequences, simple repeats, rolling circles, interspersedrepeats, small RNA, low-complexity sequences, and those sequences havingless than 100 bp in length and containing more than 3% of N were removed.After that, we excluded sequences that were present in the ArabidopsisRepBase repeats library (mostly retroelement and DNA transposon sequences)using the slow method (0%–5% more sensitive, two to three times slower thandefault) and eliminated vector and organelle sequences using the Core NCBIvector and the Arabidopsis plastid database, respectively. Once the sequenceswere masked, the ESTs were assembled and clustered using the CAP3 pro-gram included in the EGassembler service using a maximum gap length inany overlap of 20 bases, with a cutoff of more than 75% identity and anoverlap similarity score cutoff of 700. This service provided the sequences ofthe contigs fully aligned as a text file, and this was visually inspected to get arough idea about the quality of the contig sequences obtained. A similaritysearch and functional annotation were performed with the assembled se-quences containing contigs and singletons using the JAVA package Blast2Gorun under Windows (Götz et al., 2008). This was done in several consecutivesteps in an attempt to unravel the putative function of the maximum numberof sequences. In a first step, a regular BLASTX search was used using thecorresponding default NCBI-deposited protein databases with a cutoff expectvalue of 1.0E-05 for the significance similarity using the nr (for nonredundant)database located at the NCBI. All sequences that gained a BLASTX hit wereused to get additional Gene Ontology, InterPro, Enzyme, and KEGG func-tional annotations. However, for further consideration, analyses, and gener-ation of tables and graphics, we excluded those sequences with hits withe-values higher than 1.0E-15. Gene Ontology annotation was done using theGene Ontology Blast2Go database deposited in the Spanish server dated May2010. Finally, with the set of sequences lacking BLASTX hits, we conductedsearches using both BLASTN and TBLASTX separately with the only intentionof assigning them a putative candidate not present in protein databases. SinceBLASTN and TBLASTX account for DNA sequences, they could not be furtherannotated for Gene Ontology like the rest of the sequences.

Microarray Generation and Analysis

A custom-made oligonucleotide-based (60-mer length) platform repre-senting a total of 18,152 unigenes of strawberry was designed (RocheNimbleGen) from the nonredundant sequences. For each of the sequences,four oligonucleotides were printed per block, and four blocks were printedfor each data set. Total RNA samples were treated with DNase I and purifiedby Qiagen columns according to the manufacturer’s instructions. Labeling(Cy3) of samples, hybridization, and data normalization were performedaccording to the procedures described in the expression analysis sectionpublished by Roche NimbleGen (http://www.nimblegen.com/). Briefly,10 mg of total receptacle RNA was processed using the Roche cDNA Syn-thesis System. The cDNA was purified using the High Pure PCR ProductPurification Kit according to the manufacturer’s protocol. Afterward, thesamples were processed using full-size reverse transcription reactions. Threereplicate reverse transcription reactions were performed for each total RNAinput amount. Each cDNA sample was random primer labeled with Cy3nonamers according to Roche NimbleGen’s standard protocol usingNimbleGen One-Color DNA Labeling Kits. One microgram of cDNA wasused in the labeling reaction. Using random assignment, each Cy3-labeledcDNA sample was applied to the custom-made strawberry 12x135K arraysformat (each slide contains 12 independent arrays, each with 140,572 probescovering 35,143 ESTs, with four probes per target gene). The array was thenhybridized for 16 h at 42°C, washed, dried, and scanned at 2-mm resolutionusing a NimbleGen MS 200 Microarray Scanner (Roche NimbleGen). TheNimbleScan version 2.6 software (Roche NimbleGen) was used to extractfluorescence intensity signals from the scanned images and perform robustmultiarray (RMA) analysis to generate gene expression values. RMA wasperformed across replicate arrays within each test condition, sample, and




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input amount (e.g. separate RMA analyses were performed for the data setsfrom the three replicate hybridizations using cDNA derived from 10 mg oftotal RNA). Data analysis of the microarray expression studies was performedwith the DNASTAR software for gene expression analysis (http://www.dnastar.com/). The moderated Student’s t test and false discovery rate(Benjamini and Hochberg, 1995) for multiple testing corrections were usedwith a confidence of P , 0.01 to identify statistically significant differences.

Genotype and QTL Analysis

A genomic sequence of Fragaria vesca was analyzed to find the sequencearound the gene FaPRX27. A SSR was detected 50 kb upstream of the geneusing Tandem Repeat Finder software (http://tandem.bu.edu/trf/trf.html).The design of primers flanking the SSR was made using Primer 3 software(http://frodo.wi.mit.edu). The Spanish population of cultivated strawberrywas genotyped with the SSRCL317CGf marker. DNA from 93 individuals ofan F2 Dover 3 Camarosa population was extracted and amplified by PCRusing SSRCL317CGf primers (forward, 59-AGTGTGCAGTTTCCACAACG-39;reverse, 59-TGCGGAATTGATGTTCTGTC-39). SSRCL317CGf was mapped inan F2 Dover 3 Camarosa population using JOINMAP software (van Ooijen,2006). QTL detection was performed by composite interval mapping (Zeng,1994) using QTL Cartographer software (Basten et al., 1997) separately foreach parent using the female and male maps for the French pseudo test-crosssegregating population and using the linkage maps for the Spanish F2 seg-regating population. A forward-backward stepwise regression was performedto choose the five cofactors with the highest F values before performing QTLdetection by composite interval mapping. A window size of 10 centimorganaround the test interval, where the cofactors were not considered, was chosen(model 6 of QTL Cartographer). The statistical significance threshold for de-claring a putative QTL was determined by permutations of the data set. After1,000 permutations, mean log of the odds thresholds of 3.1 and 3.0 werechosen for the French and Spanish data, respectively. This corresponded to agenome-wise significance level of a = 0.05. The proportion of phenotypicvariation explained by each significant marker was estimated as the coef-ficient of determination (r2) at the peak QTL position estimated by QTLCartographer.

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession number JX290513.1.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Nucleotide sequence alignment of gene 19544,FaPRX27, and homologous sequences.

Supplemental Figure S2. Nucleotide (top) and protein (bottom) sequencesof functionally characterized FaPRX27.

Supplemental Figure S3. Nucleotide sequences of scaffold 0513171 (partialsequence), FaPRX27, and gene 19544 as predicted by the F. vesca genomesequence (Shulaev et al., 2011).

Supplemental Figure S4. SDS-PAGE analysis of four different samples ofsoluble crude protein extract obtained from FaPRX27-expressing E. coli.

Supplemental Figure S5. FaPRX27 enzyme assays with cinnamic acid.

Supplemental Figure S6. FaPRX27 enzyme assays with 4-coumaric acid.

Supplemental Figure S7. FaPRX27 enzyme assays with caffeic acid.

Supplemental Figure S8. FaPRX27 enzyme assays with ferulic acid.

Supplemental Figure S9. FaPRX27 enzyme assays with guaiacol.

Supplemental Figure S10. FaPRX27 enzyme assays with vanillin.

Supplemental Figure S11. FaPRX27 enzyme assays with coniferyl alcohol.

Supplemental Figure S12. FaPRX27 enzyme assays with cinnamaldehyde.

Supplemental Figure S13. FaPRX27 enzyme assays with 4-hydroxybenzal-dehyde.

Supplemental Figure S14. FaPRX27 enzyme assays with 3,4-dihydroxy-benzaldehyde.

Supplemental Figure S15. Description of candidate genes.

Supplemental Table S1. Strawberry varieties used for comparative micro-array analyses.

ACKNOWLEDGMENTS

We thank Aurélie Petit and Philippe Chartier from Ciref for providing thestrawberry fruits and are grateful to Miguel Angel Hidalgo from Planasa forproviding Spanish strawberry fruits.

Received June 5, 2013; accepted July 3, 2013; published July 8, 2013.

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