isolation and molecular characterisation of flavonoid 3′-hydroxylase

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UNCORRECTED PROOF 1 Short Communication 2 Isolation and molecular characterisation of avonoid 3-hydroxylase and 3 avonoid 3,5-hydroxylase genes from a traditional Chinese medicinal plant, 4 Epimedium sagittatum 5 Wenjun Q1 Huang a, b , Wei Sun b, c , Ying Wang a, 6 a Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, 430074, China 7 b Graduate University of the Chinese Academy of Sciences, Beijing, 100039, China 8 c Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, China 9 10 abstract article info 11 Article history: 12 Accepted 15 November 2011 13 Available online xxxx 14 15 16 17 Keywords: 18 Anthocyanin 19 CYP75 20 Epimedium 21 Flavonoid biosynthesis 22 The epimedii herb, a traditional Chinese medicinal plant, has signicant pharmacological effects on human 23 health. The bioactive components in the herb (Epimedium sagittatum (Sieb. et Zucc.) Maxim) are mainly 24 prenylated avonol glycosides, which are end-products of the avonoid biosynthetic pathway. This has not 25 been clearly elucidated until recently. The genes encoding avonoid 3-hydroxylase (F3H) and avonoid 26 3,5-hydroxylase (F35H) involved in the avonoid biosynthetic pathway, designated as EsF3H and EsF327 5H, were isolated from E. sagittatum using a homology-based cloning method and deposited in the GenBank 28 databases (GenBank ID: HM011054 and HM011055), respectively. EsF3H and EsF35H proteins shared high 29 homology with other plant-specic avonoid hydroxylases and were clustered into the CYP75B and CYP75A 30 group, respectively. In addition, four conserved cytochrome P450-featured motifs were found in the amino 31 acid sequences of both genes. Transcription levels of both genes were detected in all tissues tested and 32 were high in most of the pigmented tissues. Moreover, the expression levels of both EsF3H and EsF35H cor- 33 related positively with the anthocyanin accumulation pattern in leaves from E. sagittatum. The cloning and 34 molecular characterisation of EsF3H and EsF35H genes will accelerate progress in the study of the avonoid 35 biosynthetic pathway to elucidate the molecular mechanisms of the biosynthesis of the bioactive compo- 36 nents in E. sagittatum. 37 © 2011 Elsevier B.V. All rights reserved. 38 39 40 41 42 1. Introduction 43 Flavonoids represent a large class of secondary metabolites in plants 44 that are best known as the characteristic red, blue, and purple anthocy- 45 anin pigments of plant tissues (Winkel-Shirley, 2001, 2002). They have 46 a wide range of biological functions as they provide pigmentation to 47 owers, fruits, and seeds in order to attract pollinators and seed dis- 48 persers, protect plants from UV radiation, defend against phytopatho- 49 gens, act as signal molecules in plantmicrobe interactions, and are 50 involved in auxin transport and pollen germination (Dixon and Paiva, 51 1995; Koes et al., 2005; Peer and Murphy, 2007). Flavonoids receive 52 substantial public attention because of their signicant effects on 53 human health. The antioxidant activity of avonoids plays a vital role 54 in the prevention of neuronal and cardiovascular illnesses, cancer and 55 diabetes (Harborne and Williams, 2000; Havsteen, 2002). 56 The genetics and biochemistry of the avonoid pathway have been 57 characterised in several model plant species such as Arabidopsis, 58 maize, petunia, and snapdragon, and the main structural and regulatory 59 genes have been cloned (Holton and Cornish, 1995; Mol et al., 1998; 60 Winkel-Shirley, 2001). The F3H and F35H genes, both of which belong 61 to the cytochrome P450 superfamily, catalyse hydroxylation at the 362 and 3,5positions of the B-ring of the avonoids. This leads to the 63 production of the red cyanidin-based pigments and the blue/violet 64 delphinidin-based pigments, respectively (Ayabe and Akashi, 2006; 65 Tanaka, 2006). In addition to the hydroxylation of anthocyanidins, both 66 genes also catalyse the hydroxylation of avanones, avones and avo- 67 nols. Since both the F3H and F35H genes were rst isolated from petu- 68 nia (Brugliera et al., 1999; Holton et al., 1993), their homologues have 69 been subsequently isolated from many plants such as the apple (Han 70 et al., 2010), Arabidopsis (Schoenbohm et al., 2000), gentian (Tanaka et 71 al., 1996), grape (Bogs et al., 2006) and tomato (Olsen et al., 2010). Of 72 these two genes, F35H has evoked more interest from scientists and 73 industries, because some important ornamental plants, such as roses, 74 carnations and chrysanthemums lack F35H enzyme activity and cannot 75 produce blue or violet owers (Tanaka, 2006). 76 Both F3H and F35H play important roles in avonoid biosynthesis 77 and regulation. They are two of the main structural genes encoding Gene xxx (2012) xxxxxx Abbreviations: CTAB, cetyl trimethyl ammonium bromide; CYP, cytochrome P450; RACE, rapid amplication of cDNA ends; qRT-PCR, quantitative reverse transcription- polymerase chain reaction; F3H, avonoid 3-hydroxylase; F35H, avonoid 3,5- hydroxylase; FLS, avonol synthase; DFR, dihydroavonol 4-reductase. Corresponding author. Tel.: + 86 27 87510675; fax: + 86 27 87510331. E-mail address: [email protected] (Y. Wang). GENE-37119; No. of pages: 6; 4C: 0378-1119/$ see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.11.029 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Please cite this article as: Huang, W., et al., Isolation and molecular characterisation of avonoid 3-hydroxylase and avonoid 3,5-hydrox- ylase genes from a traditional Chinese medicinal plant, Epimedium sagittatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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Page 1: Isolation and molecular characterisation of flavonoid 3′-hydroxylase

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Gene xxx (2012) xxx–xxx

GENE-37119; No. of pages: 6; 4C:

Contents lists available at SciVerse ScienceDirect

Gene

j ourna l homepage: www.e lsev ie r .com/ locate /gene

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Short Communication

Isolation and molecular characterisation of flavonoid 3′-hydroxylase andflavonoid 3′, 5′-hydroxylase genes from a traditional Chinese medicinal plant,Epimedium sagittatum

Wenjun Huang a,b, Wei Sun b,c, Ying Wang a,⁎a Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, 430074, Chinab Graduate University of the Chinese Academy of Sciences, Beijing, 100039, Chinac Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong, 510650, China

Abbreviations: CTAB, cetyl trimethyl ammonium broRACE, rapid amplification of cDNA ends; qRT-PCR, quanpolymerase chain reaction; F3′H, flavonoid 3′-hydroxyhydroxylase; FLS, flavonol synthase; DFR, dihydroflav⁎ Corresponding author. Tel.: +86 27 87510675; fax:

E-mail address: [email protected] (Y. Wang).

0378-1119/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.gene.2011.11.029

Please cite this article as: Huang, W., et al.,ylase genes from a traditional Chinese med

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Article history:Accepted 15 November 2011Available online xxxx

Keywords:AnthocyaninCYP75EpimediumFlavonoid biosynthesis

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ECTED PThe epimedii herb, a traditional Chinese medicinal plant, has significant pharmacological effects on human

health. The bioactive components in the herb (Epimedium sagittatum (Sieb. et Zucc.) Maxim) are mainlyprenylated flavonol glycosides, which are end-products of the flavonoid biosynthetic pathway. This has notbeen clearly elucidated until recently. The genes encoding flavonoid 3′-hydroxylase (F3′H) and flavonoid3′, 5′-hydroxylase (F3′5′H) involved in the flavonoid biosynthetic pathway, designated as EsF3′H and EsF3′5′H, were isolated from E. sagittatum using a homology-based cloning method and deposited in the GenBankdatabases (GenBank ID: HM011054 and HM011055), respectively. EsF3′H and EsF3′5′H proteins shared highhomology with other plant-specific flavonoid hydroxylases and were clustered into the CYP75B and CYP75Agroup, respectively. In addition, four conserved cytochrome P450-featured motifs were found in the aminoacid sequences of both genes. Transcription levels of both genes were detected in all tissues tested andwere high in most of the pigmented tissues. Moreover, the expression levels of both EsF3′H and EsF3′5′H cor-related positively with the anthocyanin accumulation pattern in leaves from E. sagittatum. The cloning andmolecular characterisation of EsF3′H and EsF3′5′H genes will accelerate progress in the study of the flavonoidbiosynthetic pathway to elucidate the molecular mechanisms of the biosynthesis of the bioactive compo-nents in E. sagittatum.

© 2011 Elsevier B.V. All rights reserved.

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R1. Introduction

Flavonoids represent a large class of secondarymetabolites in plantsthat are best known as the characteristic red, blue, and purple anthocy-anin pigments of plant tissues (Winkel-Shirley, 2001, 2002). They havea wide range of biological functions as they provide pigmentation toflowers, fruits, and seeds in order to attract pollinators and seed dis-persers, protect plants from UV radiation, defend against phytopatho-gens, act as signal molecules in plant–microbe interactions, and areinvolved in auxin transport and pollen germination (Dixon and Paiva,1995; Koes et al., 2005; Peer and Murphy, 2007). Flavonoids receivesubstantial public attention because of their significant effects onhuman health. The antioxidant activity of flavonoids plays a vital rolein the prevention of neuronal and cardiovascular illnesses, cancer anddiabetes (Harborne and Williams, 2000; Havsteen, 2002).

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mide; CYP, cytochrome P450;titative reverse transcription-lase; F3′5′H, flavonoid 3′,5′-onol 4-reductase.+86 27 87510331.

rights reserved.

Isolation and molecular charaicinal plant, Epimedium sagit

The genetics and biochemistry of the flavonoid pathway have beencharacterised in several model plant species such as Arabidopsis,maize, petunia, and snapdragon, and themain structural and regulatorygenes have been cloned (Holton and Cornish, 1995; Mol et al., 1998;Winkel-Shirley, 2001). The F3′H and F3′5′H genes, both of which belongto the cytochrome P450 superfamily, catalyse hydroxylation at the 3′and 3′, 5′ positions of the B-ring of the flavonoids. This leads to theproduction of the red cyanidin-based pigments and the blue/violetdelphinidin-based pigments, respectively (Ayabe and Akashi, 2006;Tanaka, 2006). In addition to the hydroxylation of anthocyanidins, bothgenes also catalyse the hydroxylation of flavanones, flavones and flavo-nols. Since both the F3′H and F3′5′H genes were first isolated from petu-nia (Brugliera et al., 1999; Holton et al., 1993), their homologues havebeen subsequently isolated from many plants such as the apple (Hanet al., 2010), Arabidopsis (Schoenbohm et al., 2000), gentian (Tanaka etal., 1996), grape (Bogs et al., 2006) and tomato (Olsen et al., 2010). Ofthese two genes, F3′5′H has evoked more interest from scientists andindustries, because some important ornamental plants, such as roses,carnations and chrysanthemums lack F3′5′H enzyme activity and cannotproduce blue or violet flowers (Tanaka, 2006).

Both F3′H and F3′5′H play important roles in flavonoid biosynthesisand regulation. They are two of the main structural genes encoding

cterisation of flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydrox-tatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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enzymes for the flavonoid biosynthesis and function as a regulatoryfactor in an important branch point between flavonol and anthocyaninbiosyntheses. It was demonstrated in the petunia, potato and tomatothat F3′H and F3′5′H competed with flavonol synthase (FLS) and dihy-droflavonol 4-reductase (DFR) for the same substrate to determinethe branch flow (Olsen et al., 2010). In the grapevine, the transcriptionlevels of VvF3′H and VvF3′5′H were consistent with the accumulationpattern of the anthocyanins (Castellarin et al., 2006). Recently, theexpression pattern of apple F3′H genes has also been reported to corre-spond to the accumulation patterns of flavonoids in apple fruit (Han etal., 2010). Ectopic expression of F3′H and F3′5′H genes not only changedthe anthocyanin composition and content but also altered the flowercolour, such as ectopic expression of VvF3′H and VvF3′5′H in petunia(Bogs et al., 2006) and MdF3′H in tobacco (Han et al., 2010).

The epimedii herb (‘Yin-yang-huo’, used by Chinese druggists) isone of the most popular traditional Chinese medicines, with morethan two thousand years of history, and is collected from the dried ae-rial parts of the Epimedium species of Berberidaceae (Guo and Xiao,2003; Li et al., 2005). Epimedium sagittatum (Sieb. et Zucc.) Maxim,together with four other Epimedium species, Epimedium brevicornuMaxim, Epimedium brevicornu pubescens Maxim, Epimedium wusha-nense T. S. Ying, and Epimedium koreanum Nakai, is recorded in theChinese Pharmacopoeia (Pharmacopoeia Commission of PRC, 2005).Many studies have demonstrated that epimedii have extensive phar-macological efficacy for treating impotence, spermatorrhea, infertil-ity, amenorrhea, rheumatic arthritis and chronic bronchitis (Lee etal., 1995; Meng et al., 2005; Wu et al., 2003). In addition, Epimediumspecies are used as ground covers and ornamental plants due to theabundance of colours and patterns in their leaves and flowers.

It has been reported that themain bioactive constituents of epimediiare prenylated flavonol glycosides (Shen et al., 2007), which are end-products of a branch of the flavonoid biosynthetic pathway. To date,about 130 components have been identified (Wu et al., 2003), amongwhich epimedin A, B, C and icariin are consideredmajor bioactive com-ponents that comprise more than 52% of total flavonoids in herbaepimedii (Zhang et al., 2008). However, the biosynthetic pathwaysinvolved in producing these bioactive components are still unclear.

Since the 1990s, the wild resources of medicinal Epimedium spe-cies have dramatically been reduced due to years of over-harvestingand curtailment of habitat, and recently, they have even becomeendangered (Xu et al., 2008). Considering the shortage of naturalresources and the huge market demand, the creation of new cultivarsis crucial for large scale cultivation. In order to manipulate the biosyn-thesis of the bioactive components, the first step needed is to eluci-date the flavonoid biosynthetic pathway in E. sagittatum. The F3′Hand F3′5′H genes not only encode enzymes involved in the flavonoidbiosynthetic pathway but also regulate the flow orientation of theanthocyanin and flavonol pathways at an important branch point.Notably, a red-purple leaf colour has been discovered in some indi-viduals of E. sagittatum, which contrasts with the green that is usuallyobserved. Furthermore, the colour of leaves can change from red-purple to green as the developmental stage progresses. Therefore, itis possible that the EsF3′H and EsF3′5′H genes are responsible forthis colour pattern by modulating the anthocyanin accumulation pat-tern. Here, we report the isolation, molecular characterisation andexpression patterns of EsF3′H and EsF3′5′H genes in various tissuesof E. sagittatum, particularly in green and red leaves that accumulatedifferent anthocyanin contents, which shed light on the flavonoidbiosynthetic pathway in E. sagittatum.

2. Materials and methods

2.1. Plant materials

Plantlets of E. sagittatumwere collected from individuals in the ex-perimental field of Wuhan Botanical Garden that were originally

Please cite this article as: Huang, W., et al., Isolation and molecular charaylase genes from a traditional Chinese medicinal plant, Epimedium sagit

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introduced from Hunan province, China. Leaf, petiole, flower bud, flow-er, and root were sampled and immediately frozen in liquid nitrogenand kept at−70 °C until required.

2.2. Total genomic DNA and RNA extraction

Total genomic DNA from young leaveswas extracted using the CTABmethod (Doyle and Doyle, 1990). Total RNA was isolated from thevarious tissues collected above using the TRIzol® reagent (Invitrogen,USA) except roots, from which total RNA was extracted with RNAisoPlus (Takara, Japan) combined with the RNAiso-mate for plant tissue(Takara, Japan). The RNA solution was digested with RQ RNase-freeDNase I (Promega, USA) to remove any contaminating genomic DNAbefore the reverse transcription reaction.

2.3. Isolation of EsF3′H and EsF3′5′H homologues

Based on the conserved domains of the F3′H and F3′5′H genes,two pairs of degenerate primers (F3′H degF/R, F3′5′H degF/R) weredesigned to amplify the corresponding sequences in E. sagittatum(Supplemental Table S1). The partial cDNA fragments of the putativeF3′H and F3′5′H homologues were isolated by RT-PCR, using the totalRNA extracted from the young red leaves and Superscript II reversetranscriptase (Invitrogen, USA). The 439 bp and 578 bp gene specificfragments of putative F3′H and F3′5′H, respectively, were amplifiedand ligated into the pMD18-T vector (Takara, Japan). Fifteen positiveclones for each fragment were selected for DNA sequencing. Thesequence results were BLASTed against the NR protein database toidentify the putative EsF3′H and EsF3′5′H targets.

To isolate the putative EsF3′H and EsF3′5′H full-length cDNA clones,5′ RACE and 3′ RACEwere carried out using the SMARTRACEAmplifica-tion kit (Clontech, Japan). Four pairs of gene-specific primers (F3′H. 5′GSP1/2 and F3′H. 3′GSP1/2, F3′5′H. 5′GSP1/2 and F3′5′H. 3′GSP1/2)were designed to amplify the 5′ cDNA ends and 3′ cDNA ends of EsF3′H and EsF3′5′H clones, respectively (Supplemental Table S1). Accordingto the manufacturer's instructions, the single fragments for each RACEreaction were amplified and ligated into the pMD18-T vector (Takara,Japan) and sequenced.

Two pairs of gene-specific primers (F3′H. F/R, F3′5′H. F/R) weredesigned to isolate the full length cDNAs of EsF3′H and EsF3′5′H genesand the corresponding genomic sequences (Supplemental Table S1).One microliter of 5′ cDNA-to-ready template and 1 μL of genomic DNAtemplate, combined with 1.25 units of PrimeSTAR HS DNA polymerase(Takara, Japan), were used in the kit-recommended 50 μL PCR reaction.Cycling conditionswere as follows: pre-denaturation at 98 °C for 1 min,followed by 30–32 cycles of amplification (98 °C for 10 s, 55 °C for 15 sand 72 °C for 2 min), then succeeded by a final extension at 72 °C for8 min. The amplified fragments were ligated into the pMD18-T vector(Takara, Japan) and sequenced.

2.4. Sequence and phylogenetic analysis

The Blast program at the NCBI website was used to determine theputative F3′H and F3′5′H clones. The ORF finder program was used tosearch for open reading frames in the F3′H and F3′5′H nucleotidesequences. The Spidey program was used to analyse the exon andintron genomic structure. Conserved domains were searched forusing the Conserved Domain program. Multi-alignment analysis wasperformed by the ClustalW program, and the neighbour-joiningmethod by the MEGA 4.0 program was conducted to generate thephylogenetic tree from the ClustalW alignment results.

2.5. Quantitative RT-PCR for transcription levels of EsF3′H and EsF3′5′H

To detect the transcription levels of EsF3′H and EsF3′5′H genes indifferent tissues of E. sagittatum, quantitative RT-PCR was carried out.

cterisation of flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydrox-tatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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One microgram of each total RNA from leaf, petiole, flower bud, flowerand rootwas used for the reverse transcription reactionwith the Prime-script RT reagent kit combined with gDNA eraser to remove any con-taminated genomic DNA (Takara, Japan). Leaf tissues included threedifferent samples: green old leaves, red old leaves and red young leaves,which were collected from a single plant at different developmentalstages. qPCR system was recommended by the SYBR Premix Ex Taq IIkit instructions (Takara, Japan) and run by Stepone Plus equipment(ABI, USA). Gene-specific primers (F3′H.qF/R, F3′5′H.qF/R) in the qPCRassay were designed, and the actin homologue (Actin.qF/R) was usedas an internal control (Supplemental Table S1). The comparative Ctmethod was used to determine the relative expression, and the flowertissues were set as the calibrator sample (Livak and Schmittgen, 2001).

2.6. Determination of the anthocyanin content

Anthocyanins were extracted and measured as described byMancinelli (1990). Green and red mature leaves from the same E.sagittatum individual were harvested and frozen at −70 °C until re-quired. The samples (about 200–300 mg) were ground into fine pow-der with liquid nitrogen. Anthocyanins were extracted with 1% HClin methanol for 24 h at 4 °C in darkness with occasional shaking. Theextracts were centrifuged and decanted carefully, and their absorbancewas measured at 530 nm (peak absorption of anthocyanins) and657 nm (peak absorption of chlorophyll degradation products). Theequation A530-0.25A657 was used to compensate for the absorption

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Fig. 1. Comparison of the deduced amino acid sequences of E. sagittatum F3′H and F3′5′H. Numstrong similar, (.) means weak similar. Identical amino acid residues are shaded in black, simioxygen binding pocket motif, EXXR motif and heme binding domain are boxed.

Please cite this article as: Huang, W., et al., Isolation and molecular charaylase genes from a traditional Chinese medicinal plant, Epimedium sagit

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of chlorophyll degradation products at 530 nm. Anthocyanin contentwas calculated as cyanidin-3-glucoside using 29,600 as the molecularextinction coefficient and 445 as the molecular weight becausecyanidin-3-glucoside is the most common anthocyanin pigment innature (Francis and Markakis, 1989). Three independent replicateswere analysed for each sample.

3. Results and discussion

3.1. Isolation and sequence analysis of EsF3′H and EsF3′5′H clones

To obtain the F3′H and F3′5′H clones from E. sagittatum, weemployed a homology-based cloningmethod using degenerate primersdesigned in the conserveddomains of the known F3′H and F3′5′H genes.Two fragments, 439 bp and 578 bp, were amplified and sequencedas the putative EsF3′H and EsF3′5′H targets, respectively. The deducedamino acid sequence of the partial fragment EsF3′H and EsF3′5′H geneshowed 84% and 73% identities with Lobelia erinus F3′H (GenBankaccession no. BAF49324) and Vinca major F3′5′H (GenBank accessionno. ACZ63205), respectively. Only one representative candidate se-quence of each gene was found to represent the EsF3′H and EsF3′5′Hconserved domains from multiple clone sequencing results. This resultindicates that only one copy of each gene exists in the genome of E.sagittatum. This was inconsistent with the reports about their reportedcopy numbers in grapes and petunias (Brugliera et al., 1999; Castellarinet al., 2006). Therefore, further DNA gel blot analysis is needed to

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bers indicate position of amino acid from the N-terminus. (*) means identical, (:) meanslar in grey. The P450-featured conserved motif, including the proline-rich “hinge” region,

cterisation of flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydrox-tatum, Gene (2012), doi:10.1016/j.gene.2011.11.029

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determine the copy numbers in E. sagittatum. In addition, the EsF3′5′Hdegenerate primers amplified not only the target EsF3′5′H fragmentbut also the off-target EsF3′H fragment because fragment sequencesfrom EsF3′5′H clones also included the off-target EsF3′H fragmentsequences. The very high levels of identity between the conserved F3′H and F3′5′H domains, belonging to the CYP75 family, complicate thedegenerate primers design.

To obtain the full-length cDNA sequences of the EsF3′H and EsF3′5′H genes, 3′-and 5′-RACE methods were performed. The full-lengthcDNA of EsF3′H was 1700 bp containing a 1545 bp open readingframe (ORF) encoding 514 amino acids (GenBank ID: HM011054).The full-length cDNA of EsF3′5′H was 1633 bp containing a 1527 bpORF encoding 508 amino acids (GenBank ID: HM011055). Remark-ably, the EsF3′5′H gene had a very short 3′-untranslated region andno typical polyA signal sequence in the full-length cDNA sequence,which may indicate that there is another transcript that was notfound.

The deduced amino acid sequences of the EsF3′H and EsF3′5′Hgenes showed high homology to several flavonoid hydroxylases fromother species. The protein sequence encoded by EsF3′H (GenBankID: ADE80941) showed 74%, 74%, 70% identities and 86%, 85%,82% positives with other plant F3′H proteins from Malus x domestica(ACR14867), Vitis vinifera (BAE47004) and Petunia x hybrida(AAD56282), respectively, while the protein sequence encoded byEsF3′5′H (GenBank ID: ADE80942) had a high identity of 74%, 73%and high positives of 84%, 83% with other plant F3′5′H proteinsfrom V. vinifera (BAE47007) and P. x hybrida (CAA80265), respec-tively. Pair-alignment analysis showed no significant similarity be-tween the full-length nucleotide sequences of EsF3′H and EsF3′5′HcDNA, but 49% identity and 71% positives between the deducedamino acid sequences (Fig. 1).

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Fig. 2. Phylogenetic tree showing selected F3′H and F3′5′H genes from other plant speciessagittatum F3′H (ADE80941) and F3′5′H (ADE80942) are marked in diamond. The protei(ABB53383), Arabidopsis thaliana (Q9SD85), Brassica napus (ABC58723), Callistephus chinenpurea (BAD00191),Malus x domestica (ACR14867),Matthiola incana (AAG49301), Petunia x hfrom Camellia sinensis (ABA40923), Campanula medium (BAA03440), Catharanthus roseus (Gentiana triflora (BAA12735), Glycine max (AAM51564), Gossypium hirsutum (AAP31058), P(AAT34974), Vinca major (BAC97831), Vitis vinifera (CAI54277), Solanum melongena (CAA50created from the ClustalW alignment using the neighbour joining method by the MEGA 4.0bers next to the nodes are bootstrap values from 1000 replicates, and lower than 50 values

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Phylogenetic analysis was carried out using the deduced aminoacid sequences of the EsF3′H and EsF3′5′H genes with other knownplant-specific flavonoid hydroxylase proteins (Fig. 2). The phyloge-netic tree was separated into two large groups, with all F3′Hs andF3′5′Hs genes clustered in the CYP75B and CYP75A clades, respective-ly (Fig. 2). The EsF3′H and EsF3′5′H genes were placed in the basal po-sition of each clade (Fig. 2). The phylogenetic analysis suggestedthat P450 enzymes with the same function form a distinctive group.

Against the conserved domain database, the P450 superfamilysignature domain was found in both EsF3′H and EsF3′5′H sequencesat N44–K479 and P37–V497 positions, respectively (data not shown). Theproline-rich “hinge” region (P33PGPKPWP40 for EsF3′H, P37PGPKGWP44for EsF3′5′H) is thought to act as a “hinge” that is required for optimalorientation of the P450 enzyme (Werck-Reichhart and Feyereisen,2000). The motif (A/G)GX(D/E)T(T/S), which forms a binding pocketfor oxygen molecules required for catalytic activity (Chapple,1998), was present in both EsF3′H and EsF3′5′H. The absolutelyconserved EXXR motif, used to stabilise the core structure, andthe most characteristic P450 consensus sequence FxxGxRxCxG,which is located in the heme domain and responsible for carbonmonoxide-binding ability, were also found in both EsF3′H and EsF3′5′H genes (Werck-Reichhart and Feyereisen, 2000) (Fig. 1). Moreover,F3′H-specific motifs “VDVKG” and “VVVAAS” (Boddu et al., 2004) havehigh similarity counterparts at M427DVKG431 and V76IVAAS81 of EsF3′Hgene, respectively, and the characteristic motif GGEK (Brugliera et al.,1999) distinguishing F3′H from F3′5′H was present at G421GKN424

in the EsF3′H gene but not in the EsF3′5′H gene.The genomic structure of the EsF3′H and EsF3′5′H genes was ana-

lysed by pairwise aligning between the full-length cDNA sequencesand the corresponding genomic sequences. Fig. 3 showed that theEsF3′H gene consisted of three exons and two introns, consistent

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grouped into the CYP75A and CYP75B cytochrome P450 subfamily, respectively. The E.n sequences are retrieved from the GenBank database: F3′H from Antirrhinum majussis (AAG49298), Gentiana triflora (BAD91808), Glycine max (ABW69386), Ipomoea pur-ybrida (Q9SBQ9), Torenia hybrid cultivar (BAB87838), Vitis vinifera (ABH06586); F3′5′HCAA09850), Delphinium grandiflorum (AAX51796), Eustoma grandiflorum (BAA03439),etunia x hybrida hf1 (CAA80265), Petunia x hybrida ‘hf2’ (CAA80266), Verbena x hybrida155), Solanum lycopersicum (ADC80513), Solanum tuberosum (AAV85470). The tree wasprogram. The scale bar represents 0.05 amino acid substitutions per site, and the num-are not indicated.

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Fig. 3. Genomic structure of F3′H and F3′5′H in E. sagittatum. Exons are shown by a greybox, and introns by a black line. Numbers indicate the length of exon and intron. Startcodon and stop codon are shown for the entire open reading frame.

5W. Huang et al. / Gene xxx (2012) xxx–xxx

with the genomic structure of F3′H in the apple (Han et al., 2010),grape (Jeong et al., 2006) and morning glory (Hoshino et al., 2003).The EsF3′5′H genomic sequence was split into two exons by one intron,which is identical to the F3′5′H gene from V. major (Mori et al., 2004)and grape (Jeong et al., 2006) (Fig. 3). However, the F3′5′H gene inseveral Solanaceae species, including the petunia, potato and tomato,have three exons and two introns (Olsen et al., 2010).

3.2. Expression patterns of EsF3′H and EsF3′5′H in various tissues

To investigate the tissue-specific expression patterns of the EsF3′Hand EsF3′5′H genes, different tissues, including green and red old leaves,red young leaves, petioles, flower buds, flowers and roots from E. sagit-tatum, were used for quantitative RT-PCR analysis (Fig. 4a). The qPCRassay results indicated that both EsF3′H and EsF3′5′H genes wereexpressed in all tissues tested (Fig. 4b), which were in agreement

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Fig. 4. Quantitative RT-PCR analysis of EsF3′H and EsF3′5′H transcription levels in various tisanalysis. (b): Transcription levels of EsF3′H and EsF3′5′H genes in various tissues by qRT-PCRthe flower is selected as the calibration sample. (c): Comparison of anthocyanin extraction(left) and old red leaves (right). (For interpretation of the references to colour in this figur

Please cite this article as: Huang, W., et al., Isolation and molecular charaylase genes from a traditional Chinese medicinal plant, Epimedium sagit

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with the expression patterns of grape VvF3′H and VvF3′5′H (Castellarinet al., 2006). EsF3′H was strongly expressed in red-purple flower budsand red young leaves, moderately in green old leaves, flowers androots, andweakly in the red old leaf and petioles (Fig. 4b). An extremelyhigh expression level of EsF3′5′H was detected in the red young leavesand red-purple flower buds, and high expression was detected in redpetioles, all of which may accumulate large amounts of anthocyanins,and weak expression levels were detected in green and red old leaves(Fig. 4b).Moreover, both EsF3′H and EsF3′5′Hwere abundantly expressedin pigmented tissues such as red young leaves, red-purple flowerbuds and red petioles, which all contain amounts of anthocyanins,except for the low expression level of EsF3′H gene in red petioles.Therefore, it was hypothesised that the EsF3′H and EsF3′5′H genescorresponded to the accumulation pattern of anthocyanins in tissuesfrom E. sagittatum, with the exception of the low transcription level ofthe EsF3′H gene in pigmented petioles. Experiments investigatingflavonoid/anthocyanin contents and compositions in petioles may beneeded to explain this exception in future because the mRNA levelsand the ratio of F3′H and F3′5′H genes were shown to control flavonoidcomposition in several grape organs (Jeong et al., 2006).

Notably, both EsF3′H and EsF3′5′H genesweremoderately expressedin the root tissues (Fig. 4b), where the bioactive components (preny-latedflavonol glycosides) of E. sagittatum also are abundantly deposited.This result indicated that the transcription levels of EsF3′H and EsF3′5′Hmaybe correlated with the accumulation pattern of the bioactive

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sues of E. sagittatum. (a): Phenotype characteristics of seven samples used for qRT-PCRanalysis. The comparative Ct method is used to determine the relative expression, andcolours in 1% HCl acidified methanol and anthocyanin contents from old green leavese legend, the reader is referred to the web version of this article.)

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components, perhaps because both genes catalyse hydroxylation notonly of anthocyanidins but also of flavonols in E. sagittatum.

The colour of leaves from a single plant can change from red togreen as the developmental stage progresses, as the three differentleaf samples collected for qPCR analysis showed (Fig. 4a). The red pig-mented leaves accumulated more anthocyanins than the green leaves(Fig. 4c). The qPCR analysis results showed that the expression levelsof both EsF3′H and EsF3′5′H genes were more highly expressed inyoung red leaves than in old green leaves. This was especially truefor EsF3′5′H expression, but both of the genes reduced their expres-sion levels in the old red leaves, which were grown right before theturning stage for colour change; EsF3′5′H expression levels had especial-ly reduced dramatically (Fig). Biological physiology and gene expressioncould change sharply in fruits and vegetables at the turning stage ofdevelopment. VvF3′H and VvF3′5′H expression patterns both increasedto the max level in seed or skin at the véraison stage (Bogs et al., 2006;Castellarin et al., 2006). Anthocyanin contents also reduced significantlywhen leaves turned from red to green (Fig. 4c). These partly contributedto the explanation for the dramatic decrease of EsF3′5′H expressionlevels. The expression patterns of the EsF3′H and EsF3′5′H genes corre-lated positively with the anthocyanin accumulation patterns in leavesfrom E. sagittatum, which were consistent with previous reports in theapple and grape that MdF3′H, VvF3′H and VvF3′5′H were expressedhigher in red cultivars than in yellow or white cultivars (Bogs et al.,2006; Han et al., 2010).

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4. Conclusion

In summary,we isolated the gene encodingflavonoid 3′-hydroxylase(F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) from E. sagittatum usinga homology-based cloning method. Sequence analysis and molecularcharacterisation proved that EsF3′H and EsF3′5′H homologues werethe real candidate genes, belonging to the CYP75B and CYP75A sub-families, respectively. Transcription levels of the EsF3′H and EsF3′5′Hgeneswere detected in all tissues tested, and theywere highly expressedin most of the pigmented tissues. Moreover, the expression levels ofboth genes correlated positively with the anthocyanin accumulationpattern in leaves. The cloning and molecular characterisation of theEsF3′H and EsF3′5′H genes will accelerate studies about the flavonoidbiosynthetic pathway in E. sagittatum and further build an importantfoundation for genetic engineering to improve agricultural quality.

Supplementary data to this article can be found online at doi:10.1016/j.gene.2011.11.029.

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NCOAcknowledgments

Thisworkwas supported by theNationalNatural Science Foundationof China (30800624), the CAS/CAFEA International Partnership Programfor Creative Research Teams (0921101001), and the Hubei NaturalScience Foundation (2009CDA073), and Knowledge Innovation Projectof The Chinese Academy of Sciences (KSCX2-YW-N-043). Special thanksProfessor Ling Yuan for critical review of our manuscript.

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