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Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria ×ananassa)

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Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria ×ananassa)
  Characterization of major enzymes and genes involved in flavonoidand proanthocyanidin biosynthesis during fruit development instrawberry ( Fragaria  ·  ananassa )  q Joa˜o R.M. Almeida  a,1 , Eleonora D’Amico  a , Anja Preuss  b , Fabrizio Carbone  a ,C.H. Ric de Vos  c , Bettina Deiml  d , Fabienne Mourgues  a , Gaetano Perrotta  a ,Thilo C. Fischer  d , Arnaud G. Bovy  c , Stefan Martens  b , Carlo Rosati  a,* a ENEA, Trisaia Research Center, Department of Genetics and Genomics, S.S.106, km 419+500, 75026 Rotondella, MT, Italy b Philipps Universita¨ t Marburg, Institut fu¨ r Pharmazeutische Biologie, Deutschhausstrasse 17A, 35037 Marburg, Germany c Plant Research International, 6700 AA Wageningen, The Netherlands d Technical University Munich, Department of Plant Sciences, Ornamental Plants and Horticultural Plant Breeding, Am Hochanger 4,85350 Freising, Germany Received 19 March 2007, and in revised form 23 April 2007Available online 21 May 2007 Abstract The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry ( Fragaria  ·  ananassa ) by combining bio-chemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derivedanthocyanins, and a larger pool of 3 0 ,4 0 -hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes weredemonstrated by means of   in vitro  assays, with special emphasis on specificity for the biologically relevant 4 0 - and 3 0 ,4 0 -hydroxylatedcompounds. Only leucoanthocyanidin reductase showed a strict specificity for the 3 0 ,4 0 -hydroxylated leucocyanidin, while other enzymesaccepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to thesynthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown forphenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidatekey steps in the biosynthesis of strawberry flavonoid end products.   2007 Elsevier Inc. All rights reserved. Keywords:  Flavonoids; Fruit ripening; Developmental gene expression; Proanthocyanidins; Recombinant enzyme activity; Strawberry; Substrate prefe-rence Fruit attractiveness and nutritional value are importanttraits for humans and in nature, where they contribute toplant seed dispersal, thus increasing plant fitness. Polyphe-nols play a crucial role in this strategy, since they first visu-ally attract flower pollinators and then animals feeding onfruits. Moreover, they protect plants from biotic and abi-otic stresses, and provide nutritional benefits [1–3].The phenylpropanoid and flavonoid pathways (Fig. 1)have been thoroughly investigated by genetic, biochemicaland molecular studies e.g. [4 and references therein]. Thepathways are modulated by developmental and environ-mental cues through structural genes and transcription 0003-9861/$ - see front matter    2007 Elsevier Inc. All rights reserved.doi:10.1016/ q Sequence data from this work are in the Genbank database under thefollowing accession numbers. cv ‘Queen Elisa’:  FaANR , DQ664192 andDQ664193;  FaANS  , AY695817 and AY695818;  FaDFR , AY695812and AY695813;  FaFGT  , AY695815 and AY695816;  FaFHT  , AY691918and AY691919 (cDNA and gDNA, respectively). cv ‘Queen Elisa’gDNAs:  FaFLS   DQ834905 ;  FaLAR  DQ834906. cv ‘Korona’ cDNAs: FaFLS   DQ087252;  FaLAR  DQ087253. * Corresponding author. Fax +39 0835974749. E-mail address: (C. Rosati). 1 Present address: Department of Applied Microbiology, Lund Univer-sity, P.O. Box 124, S-221 00 Lund, Sweden.  ABB Archives of Biochemistry and Biophysics 465 (2007) 61–71  Fig. 1. Schematic representation of the phenylpropanoid and flavonoid pathways. Structures of metabolites which are most relevant for this work, andrelative percentage levels within each class of end-products present in ripe fruits of cv. Queen Elisa (cf. Table 1), are shown. Enzyme abbreviations: 4CL,  p -coumarate:CoA ligase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; C3H,  p -coumaroyl-CoA 3-hydroxylase; C4H, cinnamic acid4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; FGTs, flavonoid glycosyltransferases; FHT,flavanone 3 b -hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; PAL, phenylalanine ammonia-lyase.62  Joa˜o.R.M. Almeida et al. / Archives of Biochemistry and Biophysics 465 (2007) 61–71  factors (TFs) 2 , which result in a complex regulation [5 andreferences therein]. Recent works addressed the synthesis[6,7] and the oxidative polymerization [8] of flavan-3-ols, which serve for proanthocyanidin (PA) biosynthesis(Fig. 1). Many flavonoid pathway enzymes leading tomajor aglycones belong to the reductase or 2-oxoglutar-ate-/Fe 2+ -dependent dioxygenase superfamilies. Further-more, some cytochrome P450 enzymes performhydroxylation reactions at 3 0 or at 3 0 ,5 0 position of the B-ring, which are critical for flavonoid patterns [2].The Rosaceae family includes many economicallyimportant fruit crops, among which the most prominent‘‘berry’’ species belong to the genera  Fragaria  and  Rubus .Despite their octoploid level,  F.  ·  ananassa  [9–12] and itswild progenitors  F. chiloensis  and  F. virginiana  [13,14] havebeen the object of molecular studies. In ripe strawberryfruits (hereafter, strawberry receptacle will be referred toas fruit), anthocyanins are the major flavonoid compoundscompared to flavonols, flavan-3-ols and simple phenols.Information on levels and qualitative composition of fla-van-3-ol polymers PAs in strawberry is scarce and doesnot precisely describe PA levels and qualitative composi-tion [e.g. 15]. Levels of phenolic compounds have beenreported to vary as a function of genetic, environmentaland post-harvest factors [16–20]. Some strawberry flavo-noid genes and enzymes have been shown to follow atwo-phase expression pattern during fruit development[21,22] i.e., early after anthesis and in the final ripeningphase. During strawberry ripening, up-regulation of chal-cone synthase ( FaCHS  ), flavanone 3 b -hydroxylase( FaFHT  ), dihydroflavonol 4-reductase ( FaDFR ) and flavo-noid glycosyltransferase ( FaFGT  ) genes [21,23] corre-sponds to an increase in enzyme activity in fruit extracts[22], which result in anthocyanin accumulation at ripe redstage. In contrast to model species, comprehensive parallelcharacterization of structural genes and major enzymescontrolling the formation of flavonoid end products instrawberry fruits is still lacking.In this work, biochemical and molecular analyses werecombinedtoadvanceintheknowledgeofthestrawberryfla-vonoid and PA metabolism. The composition of phenoliccompounds in ripe fruits and the spatial and developmentalaccumulation of major flavonoids in strawberry fruits wereanalyzed. The activities and substrate specificities of promi-nent recombinant enzymes involved in flavonoid biosynthe-sis were studied, and the organization of correspondinggeneswaselucidated.Expressionpatternsofmainstructuralgenesofphenylpropanoidandflavonoidpathwayswereana-lyzed in different organs and during fruit ripening. Materials and methods Growing and sampling of strawberry plant material  Strawberry plants of cv ‘Queen Elisa’ were grown at Institute for FruitBreeding experimental fields in Cesena, Italy. Potted strawberry plants of cv ‘Korona’ were field-grown at Department of Plant Science, Freising,Germany. Fruit samples were collected at early green (G1, 7–10 days afteranthesis, daa), intermediate green (G2, 12–14 daa), white (W, ca. 20 daa),turning (T, ca. 25 daa) and ripe red (R, ca. 30 daa) stages for RNA andmetabolite extraction. Developed leaf, petal and root samples were col-lected for DNA and RNA extraction. All samples were immediately fro-zen in liquid nitrogen and kept at   80   C until use. Cloning of flavonoid genes Fruit cDNA sequences of flavonoid genes were obtained by RT-PCRapproaches using gene-specific primers (GSPs, listed in Suppl. Table 1S).Red fruit cDNA was used as template for all genes except leucoantho-cyanidin reductase ( FaLAR ) (G2 fruit cDNA). For anthocyanidinreductase ( FaANR ), degenerate primers designed from ANR proteinalignment (Suppl. Fig. 1S) allowed the amplification of a 686-bp fragmentwith 91% amino acid homology to  Malus ANR  (AAZ12184). Then, FaANR  GSPs were designed for 3 0 and 5 0 rapid amplification of cDNAends (RACE). Anthocyanidin synthase ( FaANS  ) and  FaFHT   fullsequences were cloned by 3 0 and 5 0 RACE with GSPs designed frompublished ESTs ( ANS  : AF041396;  FHT  : AF041385). For cloning of  FaDFR  and  FaFGT   full length coding sequences, GSPs were designedbased on available sequence information ( DFR : AF029685;  FGT  :AY575056). Flavonol synthase ( FaFLS  ) was cloned first by designingRoFLSfor and RoFLSrev primers based on a  FLS   sequence from  Rosa (AB038247), which led to the amplification of a partial 1050-bp fragment,followed by 5 0 RACE with GSPs.  FaLAR  was cloned by 3 0 RACE withGSPs designed from LAR protein alignment (Suppl. Fig. 2S). Theresulting 839-bp fragment contained an ORF of 588 nucleotides with 87%amino acid homology to  Malus LAR1  (AY830131).  FaLAR  GSPs weresuccessively designed for 5 0 RACE. Corresponding genomic sequenceswere obtained using genomic DNA (gDNA) as template in PCR reactionswith appropriate primers, or by using a PCR-based walking strategyadapted from [24]. Full cDNA sequences were cloned and sequence-veri-fied in pET15b or pYES2 expression vectors using suitable primers (Suppl.Table 1S) and  Pfu  DNA polymerase. Molecular procedures If not otherwise stated, standard molecular procedures were adopted[25]. gDNA was extracted from young leaves following a CTAB method[26]. Total RNA was extracted from fruits, leaves, petals and roots [27]. For quantitative Real Time reverse transcription PCR (qRT-PCR)experiments, first strand cDNA was synthesized from 1  l g total RNA in30  l l with oligo-d(T) 17  and Superscript III (Invitrogen, Milan, Italy),according to manufacturer’s instructions. cDNA concentration in the RTmix was quantified using a ND-1000 UV spectrophotometer (NanodropTechnologies, Wilmington, USA), and 5 ng cDNA were used for qRT-PCR experiments, carried out with GSPs (Suppl. Table 2S) designed withPrimer Express (Applied Biosystems, Monza, Italy), using an ABI 7900thermocycler (Applied Biosystems, Monza, Italy) and Platinum Sybr-Green kit (Invitrogen, Milan, Italy) according to manufacturer’s instruc-tions. An actin gene, having constant expression levels (data not shown),was used to normalize raw data and calculate relative transcript levels.Means from independent experiments were subjected to one-way ANOVA 2 Abbreviations used  : 4CL,  p -coumarate:CoA ligase; ANR, anthocyani-din reductase; ANS, anthocyanidin synthase; C4H, cinnamic acid 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR,dihydroflavonol 4-reductase; DMACA,  p -methylaminocinnamaldehyde;F3 0 H, flavonoid 3 0 -hydroxylase; Fa,  Fragaria  ·  ananassa ; FGT, flavonoidglycosyltransferase; FHT, flavanone 3 b -hydroxylase; FLS, flavonol syn-thase; FW, fresh weight; GSP, gene-specific primer; LAR, leucoanthocy-anidin reductase; LC, liquid chromatography; MS, mass spectrometry;PA, proanthocyanidin; PAL, phenylalanine ammonia-lyase; PDA, pho-todiode array; PTFE, polytetrafluoroethylene; qRT-PCR, quantitativeReal Time reverse transcription PCR; QTOF, quadrupole time of flight;TF, transcription factor. Joa˜o.R.M. Almeida et al. / Archives of Biochemistry and Biophysics 465 (2007) 61–71  63  and Tukey’s pairwise comparisons using PAST ( Automated sequencing was carried out with ABI 3700 orABI 3730 xl   (Applied Biosystems, Monza, Italy) DNA sequencers. Substrate specificity assays with recombinant enzymes Flavonoid standards and other chemicals were from TransMITFlavonoidforschung (Giessen, Germany), Phytolab (Vestenbergsgreuth,Germany), Roth (Karlsruhe, Germany), Sigma (Deisenhofen, Germany)and MBI Fermentas (St. Leon-Roth, Germany). [2- 14 C]-malonyl-CoA andUDP-[U- 14 C]-glucose were from Hartmann Analytik (Braunschweig,Germany) and Amersham Biosciences (Freiburg, Germany), respectively.Synthesis of labeled substrates is described by [28,29].Yeastheterologousexpressionexperimentswerecarriedoutasdescribed[28]. Construction of bacterial expression vector pET15b-FaFGT, trans-formation of   Escherichia coli   strain BL21, growth, induction and proteinisolation was done according to manufacturer’s description (Invitrogen).Reactionmixtures(0.5 ml)contained250–500  l gtotalprotein.Bacterialand yeast cultures with empty pET15b or pYES2, and boiled proteinextracts,wereusedascontrols.Assayconditionsofeachtestaresummarizedin Suppl. Table 3S. Reaction products were extracted twice with 200  l lEtOAc.EtOAcextractswereappliedtocelluloseTLCplatesforseparation.LAR and ANR assay extracts were quantified by HPLC-DAD (Merck-Hitachi,Darmstadt,Germany)asdescribedinRefs.[6]and[7],respectively. Chemical analyses of fruit polyphenols Flavonoids and other phenolics were extracted and analyzed essen-tially as described in [30], with some modifications. Briefly, 0.5 g of frozenstrawberry powder was extracted with 1.5 ml cold methanol containing0.1% formic acid, followed by 15 min sonication. Samples were thencentrifuged at 2500 rpm for 10 min and the supernatants were filtered over0.2  l m PTFE. Analysis was carried out using a Waters W600 system and aLuna C18 column (150  ·  4.6 mm, 3 l ; Phenomenex, Torrance, CA, USA)heated to 40   C. A 5–35% acetonitrile gradient in 0.1% trifluoroacetic acid(1 ml min  1 flow rate) was used for separation. Samples were monitoredcontinuously from 210 to 600 nm by a Waters 996 PDA detector. Datawere analyzed using Waters Empower software. Absorbance spectra andretention times of eluting peaks were compared with those of commercialstandards (Sigma, Zwijndrecht, The Netherlands; Extrasynthe`se, Genay,France; Apin Chemicals, Abingdon, UK). Dose–response curves of standards (0–50  l g ml  1 ) were determined and used to quantify thesecompounds and derivatives thereof showing similar absorbance charac-teristics in the extracts. Identity of compounds was confirmed by LC-PDA-QTOF MS and MS/MS [31]. Analysis of PA composition wascarried out by treating samples with phloroglucinol as reported [32]. Results Assessment and localization of anthocyanin and flavan-3-ol major flavonoid compounds in developing strawberry fruits The spatial and developmental accumulation of flavan-3-ols was studied in cvs ‘Queen Elisa’ and ‘Korona’ bytreating fruit sections with  p -dimethylaminocinnamalde-hyde (DMACA; Fig. 2). DMACA specifically reacts withflavan-3-ol-containing compounds to give a blue coloration Fig. 2. DMACA staining of longitudinal and transversal sections of strawberry fruits at G1 (top left), G2 (top right), white (middle left), turning (middleright) and red (bottom) stages of cv ‘Korona’. Blue coloration indicates the presence of flavan-3-ols and their derivatives in tissues. DMACA treatment of ‘Queen Elisa’ fruits produced comparable staining results (data not shown).64  Joa˜o.R.M. Almeida et al. / Archives of Biochemistry and Biophysics 465 (2007) 61–71  [33]. Fruits of both ‘Queen Elisa’ and ‘Korona’ varietiesgave a similar pattern: from W to R stage, red-orangeanthocyanin levels increased, while flavan-3-ol-associatedDMACA staining generally decreased. Interestingly,DMACA staining was spatially associated with vascular,epidermis and core fruit tissues at W stage, and mostlyrestricted to vascular tissues at T and R stages. Chemical analyses of polyphenol composition in fruits Targeted analyses of fruit methanol extracts detectedfour major classes of phenolic compounds (Table 1).Anthocyanins were the most abundant monomer com-pounds, with a total concentration of more than180 mg kg  1 berry fresh weight (FW). Pelargonidin deriva-tives were by far the major anthocyanins, among whichpelargonidin-3-glucoside and pelargonidin-3- O -malonylglucoside accounted for 80% and 14% of total anthocya-nins, respectively. Total flavonol and monomer flavan-3-ol concentration was 23 and 36 mg kg  1 FW, respectively.Quercetin and kaempferol derivatives had a balanced 3:2ratio. As to flavan-3-ols, catechin was virtually the onlyaglycone present: epicatechin was detected only in traceamounts, as well as 4 0 -hydroxylated afzelechin, whose massspectrometry signal was about 25-fold lower than that of catechin in ‘Queen Elisa’ (data not shown). Phloroglucinoltreatment [32] revealed an unprecedented large flavan-3-olpool in the form of oligomeric and polymeric PAs. PA ter-minal units predominantly consisted of catechin units (10-fold more than epicatechin). In contrast, epicatechin levelas extension unit was 4-fold that of catechin (Table 1).Sugar or acyl substitutions were all at the 3 position of identified flavonoids. Flavonols were present as either glu-cosides or glucuronides, while a more complex pattern wasobserved for anthocyanins, with mono- (glucose, arabi-nose) or disaccharide (rutinose) and acylated (malonylated)substitutions of pelargonidin and only small amounts of cyanidin glucosides, in addition to three unidentifiedpelargonidin derivatives (Table 1). Finally,  p -coumaricand ellagic acid derivatives were present in relatively highamounts in red fruits, while levels of chlorogenic acid deriv-atives were below 1 mg kg  1 (data not shown). Recombinant enzyme assays Full coding sequences of   FaANR ,  FaANS  ,  FaDFR , FaFGT  ,  FaFHT  ,  FaFLS   and  FaLAR  were expressed in E. coli   or  Saccharomyces cerevisiae , respectively usingpET15b (FaFGT) or pYES2 (other enzymes) vectors (seeMethods). Cell extract preparations were obtained fromtransformed bacterial or yeast cultures after induction of protein expression, and standard assays for each enzymewere developed (Suppl. Table 3S). A number of physiolog-ically relevant substrates according to the analytical data(Table 1 and Suppl. Table 3S) were assayed for each enzyme. All expressed flavonoid genes produced functionalenzymes with the assigned activity (Table 2). In particular,4 0 -hydroxylated flavonoids were the preferred substrates of FaANS, FaDFR, FaFHT and FaFLS, whereas FaLARand FaANR showed a higher activity on 3 0 ,4 0 -hydroxyl-ated compounds. A strict substrate specificity was foundonly for FaLAR, which used only leucocyanidin as sub-strate. Combined FaDFR + FaANS enzyme assays usingdihydrokaempferol as substrate yielded both pelargonidinand kaempferol (Fig. 3): this demonstrated not only thepostulated activity of both enzymes (conversion of dihydrokaempferol into pelargonidin  via  the leucopelarg-onidin intermediate), but also FaANS side reactions [34].Other qualitative assays on unnatural substrates showedminor flavanone reductase activity of FaDFR, and FaLARactivity on 3-deoxyleucoanthocyanidins (data not shown).Concerning glycosylation of flavonoid end products,FaFGT displayed a strong 3- O -glucosyltransferase activityon both tested anthocyanidins, while flavonols seem to beonly a minor substrate (Table 2). Competition experimentsproviding various substrates gave similar ratios of productformation as those obtained from standard assays (datanot shown). Molecular characterization of flavonoid structural genes Genomic sequences were obtained by PCR with GSPsusing gDNA of ‘Queen Elisa’ as template (see Materialsand methods). Comparison between cDNA and gDNAsequences revealed the presence of introns in all genes(Suppl. Fig. 3S): one in  FaANS   and  FaFGT  ; two in  FaFHT  and  FaFLS  ; four in  FaLAR  and  FaANR ; and five in FaDFR . The number and position of gene introns weregenerally conserved when compared with those of otherspecies. Intron length varied between 94 bp (intron I of  FaDFR ) and 1383 bp (intron I of   FaFLS  ). Length poly-morphisms were found between introns of   FaANS  ,  FaDFR and  FaFHT   genes and the corresponding introns of genehomologs in  F. vesca  [35]. Amino acid sequence analysisrevealed several catalytic domains for all enzymes (Suppl.Fig. 3S). For instance, FaFGT contains the PSPG box,critical for substrate binding [36]. FaLAR has the RFLP,ICCN and THD motifs (named after the codes of ‘‘signa-ture’’ amino acids) specific of LAR proteins [37], as wellas a Gly-rich NAD(P)H-binding site [38] starting at G20.Southern analyses (data not shown) detected a variablenumber of bands for each above-studied gene in ‘QueenElisa’: one for  FaFGT  ; up to two for  FaANR ; up to 4–5for  FaANS  ,  FaDFR ,  FaFHT   and  FaLAR ; and as manyas eight for  FaFLS  . Gene expression analyses qRT-PCR experiments were carried out to determinegene transcript levels in various organs and developingfruits. In order to perform a more complete analysis, phen-ylalanine ammonia-lyase ( FaPAL ), cinnamic acid4-hydroxylase ( FaC4H  ),  p -coumarate:CoA ligase ( Fa4CL ),four  CHS   ( FaCHS1 ,  FaCHS2 ,  FaCHS3 , and  FaCHS5 ), Joa˜o.R.M. Almeida et al. / Archives of Biochemistry and Biophysics 465 (2007) 61–71  65
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