research article cloning and characterization of a...

Research ArticleCloning and Characterization of a Flavonol Synthase Gene fromScutellaria baicalensis

Yeon Bok Kim1 KwangSoo Kim2 YeJi Kim1 Pham Anh Tuan1 Haeng Hoon Kim3

Jin Woong Cho1 and Sang Un Park1

1 Department of Crop Science Chungnam National University 99 Daehak-ro Yuseong-gu Daejeon 305-764 Republic of Korea2Department of Biochemistry and Molecular Biology Baylor College of Medicine One Baylor Plaza Houston TX 77030 USA3Department of Well-Being Resources Sunchon National University 413 Jungangno Suncheon Jeollanam-do 540-742Republic of Korea

Correspondence should be addressed to Jin Woong Cho jwchocnuackr and Sang Un Park suparkcnuackr

Received 24 August 2013 Accepted 24 October 2013 Published 28 January 2014

Academic Editors J Jakse and E Porceddu

Copyright copy 2014 Yeon Bok Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Flavonols are themost abundant of all the flavonoids and play pivotal roles in a variety of plantsWe isolated a cDNA clone encodingflavonol synthase from Scutellaria baicalensis (SbFLS) The SbFLS cDNA is 1011 bp long encodes 336 amino acid residues andbelongs to a family of 2-oxoglutarate-dependent dioxygenases The overall structure of SbFLS is very similar to that of Arabidopsisthaliana anthocyanidin synthase (AtANS) with a 120573 jelly-roll fold surrounded by tens of short and long 120572-helices SbFLS wasconstitutively expressed in the roots stems leaves and flowers with particularly high expression in the roots and flowers SbFLStranscript levels in the roots were 376- 70- and 25-fold higher than in the leaves stems and flowers The myricetin content wassignificantly higher than that of kaempferol and quercetin Therefore we suggest that SbFLS mediates flavonol formation in thedifferent organs of S baicalensis Our study may contribute to the knowledge of the role of FLS in S baicalensis

1 Introduction

Scutellaria baicalensis Georgi (Lamiaceae) is one of the mostpopular herbs in several oriental countries where it is usedto treat inflammation respiratory tract infections diarrheadysentery jaundiceliver disorders hypertension hemorrhag-ing and insomnia [1] Scutellaria is amild relaxant that affectsthe neural and muscular-skeletal systems [2] S baicalensisroot has abundant flavones a class of plant flavonoids [3]

Polyphenols synthesized via the phenylpropanoid path-way can be classified into (iso)flavonoids coumarins au-rones condensed tannins and anthocyanins [4] Flavonoidsare important secondary metabolites derived from malonyl-CoA and the aromatic amino acid phenylalanine [5] approx-imately 8000 different flavonoid compounds have been iden-tified [6] Flavonols are the most abundant of all the flavon-oids [7] and play pivotal roles in a variety of plants [8]Flavonols are particularly well known for their antiox-idant anti-inflammatory antiangiogenic antiproliferativeand neuropharmacological properties and thereby account

for the health-promoting effects of grapes and manyother fruits [8 9] Flavonols are synthesized from dihy-drokaempferol (DHK) dihydroquercetin (DHQ) or dihy-dromyricetin (DHM) by flavonol synthase (FLS) enzymes(Figure 1) FLS belongs to a growing family of 2-oxoglutarate-dependent dioxygenase (2-ODD) nonheme ferrous enzymesthat also includes the flavonoid enzymes flavanone-3120573-hy-droxylase (F3H) flavone synthase Ι (FS Ι) and leucoan-thocyanidin reductase (LDOX) [10] FLS activity was firstcharacterized in irradiated parsley cells [11]Thefirst FLS genewas isolated from Petunia hybrida and expressed in yeast [12]FLS genes have recently been cloned and characterized inPopulus tremula [13] Zea mays [14] Fagopyrum tataricum[15] and Ginkgo biloba [16] Cloning and characterization ofFLS from S baicalensis have not been reported

In this study a full-length cDNA encoding FLS was iso-lated from S baicalensis by using next-generation sequencingplatforms (NGS) (Roche 454 FLX+ and IlluminaHiSeq 2000)(unpublished data)ThemRNA transcript levels and flavonolaccumulation from different organs of S baicalensis were

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 980740 7 pageshttpdxdoiorg1011552014980740

2 The Scientific World Journal

PhenylalaninePALC4H4CL

Naringenin

Chalcone

CHS

CHI

F3H

DihydroquercetinDihydrokaempferol Dihydromyrcetin

FLS FLSFLS

OOH

O

O

O

O

OKaempferol Quercetin Myricetin

Flavonols

OHOH

OH

OH

OHOH

OH

OH OH

OHOH

OH

OHOH

4-coumaroyl-CoA + 3 malonyl-CoA

F3998400H

F39984005998400H F39984005998400H

Figure 1 Flavonol biosynthesis in plants (redrawn from [37]) The red letter and black box indicate the enzyme and compound analyzed inthis study PAL phenylalanine ammonia lyase C4H cinnamate 4-hydroxylase 4CL 4-coumaroyl CoA ligase CHS chalcone synthase CHIchalcone isomerase F3H flavone 3-hydroxylase F31015840H flavonoid 31015840-hydroxylase F3101584051015840H flavonoid 3101584051015840-hydroxylase FLS flavonol synthase

analyzed by real-time PCR and high-performance liquid chr-omatography (HPLC) The cloning and characterization ofSbFLS may provide a foundation to elucidate the mechanismof flavonol synthesis in S baicalensis

2 Materials and Methods

21 Plant Material and Growth Conditions The seeds of Sbaicalensis were sown in May 2012 and then seedlings weretransferred into pots filled with perlite-mixed soil Seedlingswere grown under 16 h light and 8 h dark conditions in thegreenhouse (25∘C and 50 humidity) at ChungnamNationalUniversity (Daejeon Korea) We used at least nine pots forbiological repeats Each organ (flowers stems leaves androots) was collected for two weeks after the first day of flow-ering All samples were frozen in liquid nitrogen upon collec-tion and stored at minus80∘C prior to RNA isolation and HPLC

22 Cloning of the cDNA Encoding Flavonol Synthase A put-ative flavonol synthase was obtained from S baicalensis bynext-generation sequencing (NGS) (Roche 454 FLX+ andIllumina HiSeq 2000) (unpublished data) We obtained1226938 reads from Roche 454 FLX+ and 161417646 readsfrom Illumina HiSeq 2000 The de novo assembly of the 454and Illumina high-quality reads (95 and 75) were resultedmore than 82Mb sequence lengthwith an average contig readlength of 1614 bp in 51188 contigs which is the long lengthcontigs with at least 500 bp Among these contigs a total of39581 contigs were annotated by BLASTX against the publicNCBI nonredundant database In particular putative SbFLS

sequence was obtained by BLASTX for comparison Theprimers for the open reading frame (ORF) were as followsforward 51015840-ATGGAGGTTGGGAGAGTG-31015840 reverse 51015840-TCACTGGGGAAGCTTATTAAGCTTAC-31015840 The TM Cal-culator program (httpbioinfouteeprimer3-040) wasused to compute the PCR annealing temperatures The PCRprogram consisted of denaturation at 94∘C for 1min anneal-ing at 45∘C for 1min and an extension at 72∘C for 1minThirty cycles were preceded by denaturation step at 94∘Cfor 3min followed by a final extension at 72∘C for 10minThe amplified product was purified and cloned into the T-blunt vector (SolGent Daejeon Korea) and sequenced Thefull-length FLS sequence was aligned using BioEdit SequenceAlignment Editor version 509 [17] For putative conserveddomains we used a GenBank conserved domain databasesearch and function analysis service in NCBI

23 Total RNA Extraction and Quantitative Real-Time RT-PCR Total RNA was isolated from S baicalensis organs withanRNeasy PlantMini Kit (Qiagen Valencia CAUSA) First-strand cDNA was synthesized from total RNA (1 120583g) withReverTra Ace-120572- (Toyobo Osaka Japan) and oligo (dT)

20

primer according to the manufacturerrsquos protocol ORF andgene-specific primers were designed using an onlineprogram (httpfrodowimiteduprimers3) The gene-spe-cific primer sets were designed for SbFLS-RT (forward)51015840-ACCCAACGAGGTTCAAGGAC-31015840 and SbFLS-RT(reverse) 51015840-TGGTGGGGTCTCTTCATTCA-31015840 Real-timePCR was performed in a 20 120583L reaction volume with 05120583Meach primer and 2times SYBR Green real-time PCR master mix

The Scientific World Journal 3

(Toyobo Osaka Japan) Cycling conditions were as follows 1cycle of 95∘C for 3min followed by 40 cycles of 95∘C for 15 s72∘C for 20 s and annealing at 50∘C Real-time RT-PCR wascarried out in triplicate on a CFX96 real-time PCR system(Bio-Rad Hercules CA USA) The actin gene (GenBankaccession number HQ847728) was used as an internalreference to standardize the cDNA template concentration

24 Bioinformatic Analysis of Flavonol Synthase The de-duced amino acid sequence of SbFLS was aligned using theBioEdit Sequence Alignment Editor version 509 (Depart-ment of Microbiology North Carolina State UniversityRaleigh NCUSA) [17]Theoreticalmolecular weights (MW)and isoelectric points (pI) were calculated using the ComputepIMw tool (httpcaexpasyorgtoolspi toolhtml) Put-ative target localization of SbFLS was predicted by usingWoLF PSORT (httppsorthgcjpformhtml) [18] The phy-logenetic tree was constructed using an online program(httpwwwphylogenyfr) [19] Secondary structure predic-tion was investigated by network protein sequence ana-lysisat SOPMA (httpnpsa-pbilibcpfrcgi-binnpsa automatplpage=NPSAnpsa sopmahtml) [20] The three-dimen-sional (3D) structure was built using the SWISS-MODELprogram and illustrated with the PyMOL viewer

25 HPLC Analysis Flavonol content was determined as de-scribed in the studies by Li et al [15] and Kovacs et al [21]with a modification Flavonol standards kaempferol quer-cetin and myricetin were purchased from Sigma-AldrichThe harvested organs (flowers stems leaves and roots) werefreeze-dried for 48 h and ground into a fine powder using amortar andpestle Powdered samples (100mg)were extractedby sonication in 80 (vv) methanol at room temperature for60min During extraction samples were vortexed every15min After extraction the extracts were centrifuged at14000 rpm for 10min and the supernatant was filtered with a045 120583mAcrodisc syringe filter (Pall Corp Port WashingtonNY) for HPLC HPLC was performed with a C18 column(250 times46mm 5120583m RStech Daejeon Korea) The mobilephase was a gradient prepared from mixtures of methanoland 01 acetic acid The flow rate was maintained at07mLmin An injection volume of 20 120583L and wavelength of270 nm were used for detection The compounds in the sam-ple were determined using a standard curve Determinationswere performed after 3 separate extractions of each sampleand each extract was analyzed in triplicate (119899 = 3)

3 Results and Discussion

31 Cloning and Sequencing Analysis of FLS Based on Sbaicalensis NGS data (unpublished) we designed primersspecific to the open reading frame (ORF) and flower cDNAto generate a 1011 bp full-length fragment for use in PCRTheSbFLS amino acid sequence obtained from the PCR frag-ment contained 1 more residue than indicated by the NGSdata (data not shown) SbFLS (GenBank accession noKC404852)was composed of 336 amino acidswith aMWandpI of 383 kDa and 551 respectivelyThis result corresponded

02

CnFLSSbFLS

AmFLSStFLS

NtFLSLjFLS

GtFLSRhFLS

CuFLSRcFLS

VvFLS

Figure 2 Phylogenetic relationships of FLS protein from Sbaicalensis and selected species GenBank accession numbersare as follows NtFLS (Nicotiana tabacum ABE28017) AmFLS(Antirrhinum majus ABB53382) CnFLS (Camellia nitidissimaADZ28516) StFLS (Solanum tuberosum ACN81826) GtFLS (Gen-tiana triflora BAK09226) VvFLS (Vitis vinifera BAE75809) RhFLS(Rudbeckia hirta ABN79672) CuFLS (Citrus unshiu BAA36554)LjFLS (Lonicera japonica AFJ44313) RcFLS (Ricinus communisXP 002513772) and SbFLS (S baicalensis KC404852)

with the published sequence from Arabidopsis [22] G bilobaFLS has a 1023 bp ORF encoding a 340 amino acid proteinMW of 387 kDa and pI of 575 [16] The deduced SbFLSshared 77 74 72 72 71 and 70 identity with FLS proteinsfrom Antirrhinum majus (ABB53382) Nicotiana tabacum(ABE28017) Camellia nitidissima (ADZ28516) Gentiana tri-flora (BAK09226) Solanum tuberosum (ACN81826) andVitis vinifera (BAE75809) respectively (SupplementaryFigure S1 available online at httpdxdoiorg1011552014980740) Owens et al [23] reported that the genome of Ara-bidopsis thaliana contains 5 sequences with high similarityto AtFLS1 a previously characterized flavonol synthase gene4 AtFLS isoforms are located on chromosome 5 A GenBankconserved domain database search and function analysisrevealed that SbFLS has putative conserved domains belong-ing to the 20G-FeII Oxy super family and the highlyconservedN-terminal region of proteinswith 2-oxoglutarateFe(II)-dependent dioxygenase activityThis is consistent withthe results reported by Xu et al [16] Moreover SbFLS hadconserved sequencemotifs including the HXDmotif [15] forligating ferrous iron and the RXS motif for binding 2-oxoglutarate (2OG) [16] To determine the relationship bet-ween the putative SbFLS protein and other plant FLSs we per-formed phylogenetic analysis (Figure 2) The SbFLS phy-logeny was clustered into 2 distinct groups and showed theclosest relationship with A majus The subcellular targetingof SbFLSwas predicted by PSORT to be nuclear and cytosolicAtFLS1 localizes to the cytoplasm and nucleus [22] whereasmaize FLS localizes in the ER and perinuclear region [24]SOPMA [20] indicated that SbFLS contains 121 (36) alpha-helices 61 (186) extended strands 22 (66) beta turnsand 132 (393) random coils respectively

32 Modeled 3D-Structure of SbFLS To identify thecatalytic residues SbFLS was homologously modeled onthe 175 A resolution structure of A thaliana anthocyanidin


D224

H278

H222

QUE

E296

F294

2OG

Y207

R288

(a)

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

11111

7085696979

162178161161171

254270253253264

1205721 1205781 1205731 1205722

1205732 1205723 1205724 1205733 1205734 1205782 1205783

1205725 1205726 1205735 1205736 1205737 1205738 1205739

12057310 12057311 12057312 12057314120573131205727 1205784 1205728

(b)

Figure 3 The structural model of SbFLS (a) The overall and detailed views of the structure of SbFLS showing the 120573 jelly-roll fold andpresumed catalytic site are modeled using SWISS-MODEL server (httpswissmodelexpasyorg) [27] Iron atom is represented by a red dotSubstrates quercetin (QUE) and 2-oxoglutarate (2OG) are indicated in yellow and greenThe iron QUE and 2OG are positioned by the samestructure identified in the AtANS complex (PDB code 1GP6) Possible hydrogen bonds are indicated by dashed lines (b) Sequence alignmentof AtANS and various FLSs from S baicalensis (SbFLS) V vinifera (VvFLS) C unshiu (CuFLS) and A thaliana (AtFLS) The secondarystructure was drawn from the modeled structure of SbFLS Residues coordinating with an iron atom are represented by blue ovals Greentriangles and red rectangles indicate residues that interact with 2OG andQUE respectivelyThe figure was produced using the ESPript server(httpespriptibcpfrESPriptESPript) [38]

synthase (AtANS PDB 1GP6) [25] The overall structureof SbFLS and AtANS is very similar with a 120573 jelly-roll foldsurrounded by tens of short and long 120572-helices (Figure 3(a))The iron metal may be ligated with a bidentate group ofcosubstrate 2OGaswell as 3 side-chain residues that isH222D224 and H278 which are conserved in AtANS and SbFLS(Figures 3(a) and 3(b)) [15 25] Hydrogen bonds may formbetween the other side of 2OG and 3 conserved residues thatis Y207 R288 and S290 (Figure 3(b)) [15 25 26] A substratequercetin (QUE) might be located near an iron metal ion inwhich 5 invariant residues H133 F135 K203 F294 and E296may contribute to binding stability by forming a 120587-stackingarrangement (especially F294) with the QUE ring and by

hydrogen bonding All interactions as shown here wereless than 35 A distant Kinetic analyses of AtANS revealedthat most of the highly conserved residues play key roles insubstratecosubstrate binding and enzymatic catalysisthrough metal coordination [27]

33 Transcript Levels of SbFLS in Different Tissues Expres-sion of SbFLS was investigated in the flowers leaves stemsand roots of S baicalensis by qRT-PCR (Figure 4) Transcrip-tion of SbFLS was highest in the roots and lowest in the lea-ves SbFLS transcription in roots was 376- 70- and 25-foldhigher than in leaves stems and flowers Very recently Xu etal [16] reported that the highest level of G biloba FLS occurs


0

50

100

150

200

250

Root Stem Leaf Flower

Gen

e exp

ress

ion

relat

ive t

o ac

tin

Figure 4 Transcript levels of SbFLS in different organs The heightof each bar and error bars represent means and standard errorsrespectively from 3 independent measurements

0

03

06

09

12


Kaempferol

(mg

g dr

y w

t)

(a)

0

03

06

09

12

15


(mg

g dr

y w

t)

Quercetin

(b)

0

2

4

6

8


Myricetin

(mg

g dr

y w

t)

(c)

Figure 5 Flavonol content in different organs of S baicalensisTheheight of each bar and error bars represent the means and standarderrors respectively from 3 independent measurements

inmature leaves whereas the lowest level was observed in theroots Expression ofAtFLS isoforms follows different patternsin different tissues [23] The transcription of AtFLS1 washighest in floral buds flowers and siliques whereas the rootsand shoots of young seedlings and the lea-ves of later vegeta-tive stages exhibited lower expression Other AtFLS isoformswere expressed at much lower levels or were undetectable inall tissues [23] Therefore Owens et al [23] suggested thatonly AtFLS1 contributes to flavonol synthesis in ArabidopsisTranscription of Citrus unshiu FLS was higher in youngleaves than in old leaves and increased in the peel duringfruit maturation [28] Based on the transcription patterns inseveral plants we conclude that FLS is differentially regulatedin different plants and plant tissues

34 Accumulation of Flavonol in Different Tissues Kaemp-ferol quercetin andmyricetin were detected in almost all tis-sues (ie flowers stems leaves and roots) by HPLC(Figure 5) The myricetin content was significantly higherthan that of kaempferol and quercetinThe transcription pat-tern of SbFLS was similar to the accumulation pattern ofkaempferol Roots showed the highest kaempferol content(097mg gminus1 dry weight (DW)) the leaves and flowers con-tained similar amounts (062 and 051mg gminus1 DW) but nokaempferol was detected in the stem Kaempferol and someglycoside derivatives have a wide range of pharmacologicalactivities [29] and account for the utility of dry Scutellariaroot as a multipurpose herb in oriental medicine The leavescontained the highest levels of quercetin (126mg gminus1 DW)whereas the lowest amount of quercetin was in the flowers(064mg gminus1 DW) Interestingly roots displayed the lowestamount of myricetin (08mg gminus1 DW) whereas the highestmyricetin contentwas found in the flowers (605mg gminus1DW)The myricetin content in the flowers was 22- and 75-foldhigher than that in the leaves and roots respectively Feng andLiu [30] reported that the quercetin content of leaves was sig-nificantly higher than the content in pseudostems in differenttissues of Welsh onion (Allium fistulosum L)

Quercetin kaempferol morin rutin andmyricetin act asantioxidants and possess anti-inflammatory antiallergic anti-viral and anticancer properties [31] Quercetin is a free radi-cal scavenger that protects against liver reperfusion ischemictissue damage [32] The scavenging activity of flavonols de-creases as follows myricetin gt quercetin gt kaempferol [33]

SbFLS transcription was the highest in the roots correlat-ing with kaempferol content in this organThus SbFLSmightregulate the biosynthesis of kaempferol in S baicalensis Un-like kaempferol quercetin and myricetin were most highlyconcentrated in the leaves and flowers respectively Theexpression level of SbFLSwas not consistent with the contentsof myricetin and quercetin Owens and his colleagues [23]reported that Arabidopsis uses different isoforms of FLS withdifferent substrate specificities to mediate the production ofthe quercetin and kaempferol in different tissue or cell typesIn addition Lillo et al [34] described that FLS activity of theANS enzymemay contribute to the differential accumulationof kaempferol and quercetin Moreover it was pointed outthat differential expression of the F31015840H enzyme could also


mediate kaempferol and quercetin ratio in petunia flowers[35] Stracke et al [36] reported that FLS1 could be acti-vated by flavonol-specific transcription factors (TFs) MYB11MYB12 and MYB111 of A thaliana and these TFs caused dif-ferent spatial accumulation of specific flavonol derivatives inleaves stems inflorescences siliques and roots [23] Severalisoforms of FLS have been isolated in other plants such asArabidopsis [23] and maize [24] Therefore we presume thatSbFLS isoforms SbANS and SbF31015840H may be contributed tothe biosynthesis of quercetin and myricetin

4 Conclusion

We have described the molecular cloning and characteriza-tion of a S baicalensis gene encoding SbFLS Our results sug-gest that SbFLS is a key potential target for the flux of theflavonol biosynthesis pathway in S baicalensis To explainadequately the flavonol biosynthesis mechanisms in S bai-calensis in vitro enzyme assay of SbFLS isoforms SbANS andSbF31015840H should be examined in the near future Our studymay help to determine the role of FLS and facilitatemetabolicengineering of flavonol biosynthesis in S baicalensis

Abbreviations

HPCL High-performance liquid chromatographyCGA Chlorogenic acidPAL Phenylalanine ammonia-lyaseC4H Cinnamic acid 4-hydroxylase4CL 4-coumaroylCoA-ligaseCHI Chalcone isomeraseF3H Flavone 3-hydroxylaseF31015840H Flavonoid 31015840-hydroxylaseF3101584051015840H Flavonoid 3101584051015840-hydroxylaseFLS Flavonol synthaseANS Anthocyanidin synthase

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work (K13101) was supported by the Korea Instituteof Oriental Medicine (KIOM) Grant funded by the Koreangovernment

References

[1] M Li-Weber ldquoNew therapeutic aspects of flavones the anti-cancer properties of Scutellaria and its main active constituentsWogonin Baicalein and Baicalinrdquo Cancer Treatment Reviewsvol 35 no 1 pp 57ndash68 2009

[2] D Y Zhang JWu F Ye et al ldquoInhibition of cancer cell prolifer-ation and prostaglandin E2 synthesis by Scutellaria baicalensisrdquoCancer Research vol 63 no 14 pp 4037ndash4043 2003

[3] S Martens and A Mithofer ldquoFlavones and flavone synthasesrdquoPhytochemistry vol 66 no 20 pp 2399ndash2407 2005

[4] B Winkel-Shirley ldquoFlavonoid biosynthesis A colorful modelfor genetics biochemistry cell biology and biotechnologyrdquoPlant Physiology vol 126 no 2 pp 485ndash493 2001

[5] B W Shirley W L Kubasek G Storz et al ldquoAnalysis of Ara-bidopsismutants deficient in flavonoid biosynthesisrdquo Plant Jour-nal vol 8 no 5 pp 659ndash671 1995

[6] F Ververidis E Trantas C Douglas G Vollmer G Kret-zschmar and N Panopoulos ldquoBiotechnology of flavonoids andother phenylpropanoid-derived natural products Part I chem-ical diversity impacts on plant biology and human healthrdquo Bio-technology Journal vol 2 no 10 pp 1214ndash1234 2007

[7] H A Stafford Flavonoid Metabolism CRC Press Boca RatonFla USA 1990

[8] B H Havsteen ldquoThe biochemistry and medical significance ofthe flavonoidsrdquo Pharmacology andTherapeutics vol 96 no 2-3pp 67ndash202 2002

[9] E Butelli L TittaMGiorgio et al ldquoEnrichment of tomato fruitwith health-promoting anthocyanins by expression of selecttranscription factorsrdquo Nature Biotechnology vol 26 no 11 pp1301ndash1308 2008

[10] L Britsch J Dedio H Saedler and G Forkmann ldquoMolecu-lar characterization of flavanone 3120573-hydroxylases Consensussequence comparisonwith related enzymes and the role of con-served histidine residuesrdquo European Journal of Biochemistryvol 217 no 2 pp 745ndash754 1993

[11] L Britsch W Heller and H Grisebach ldquoConversion of flavan-one to flavones dihydroflavonol and flavonol with an enzymesystem from cell cultures of parsleyrdquo Zeitschrift Fur Natur-forschung vol 36 pp 742ndash750 1981

[12] T A Holton F Brugliera and Y Tanaka ldquoCloning and expres-sion of flavonol synthase from Petunia hybridardquo Plant Journalvol 4 no 6 pp 1003ndash1010 1993

[13] B-G Kim E J Joe and J-H Ahn ldquoMolecular characterizationof flavonol synthase from poplar and its application to the syn-thesis of 3-O-methylkaempferolrdquo Biotechnology Letters vol 32no 4 pp 579ndash584 2010

[14] M L F Ferreyra S Rius J Emiliani et al ldquoCloning and charac-terization of a UV-B-inducible maize flavonol synthaserdquo PlantJournal vol 62 no 1 pp 77ndash91 2010

[15] C Li Y Bai S Li et al ldquoCloning characterization and activityanalysis of a flavonol synthase gene FtFLS1 and its associationwith flavonoid content in tartary buckwheatrdquo Journal of Agri-cultural and Food Chemistry vol 60 pp 5161ndash5168 2012

[16] F Xu L Li W Zhang et al ldquoIsolation characterization andfunction analysis of a flavonol synthase gene from GinkgobilobardquoMolecular Biology Reports vol 39 no 3 pp 2285ndash22962012

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] P Horton K-J Park T Obayashi et al ldquoWoLF PSORT proteinlocalization predictorrdquo Nucleic Acids Research vol 35 ppW585ndashW587 2007

[19] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 pp W465ndashW469 2008

[20] C Geourjon and G Deleage ldquoSOPMA significant improve-ments in protein secondary structure prediction by consensusprediction frommultiple alignmentsrdquoComputer Applications inthe Biosciences vol 11 no 6 pp 681ndash684 1995


[21] G Kovacs I N Kuzovkina E Szoke and L Kursinszki ldquoHPLCdetermination of flavonoids in hairy-root cultures of Scutellariabaicalensis Georgirdquo Chromatographia vol 60 pp S81ndashS852004

[22] MK Pelletier J RMurrell andBW Shirley ldquoCharacterizationof flavonol synthase and leucoanthocyanidin dioxygenase genesin Arabidopsis further evidence for differential regulation ofldquoearlyrdquo and ldquolaterdquo genesrdquo Plant Physiology vol 113 no 4 pp1437ndash1445 1997

[23] D K Owens A B Alerding K C Crosby A B Bandara J HWestwood and B S J Winkel ldquoFunctional analysis of a pre-dicted flavonol synthase gene family inArabidopsisrdquo Plant Phys-iology vol 147 no 3 pp 1046ndash1061 2008

[24] F M L Falcone M I Casas J I Questa et al ldquoEvolution andexpression of tandem duplicated maize flavonol synthasegenesrdquo Frontiers Plant Science vol 3 article 101 2012

[25] R C Wilmouth J J Turnbull R W D Welford I J Clifton AG Prescott and C J Schofield ldquoStructure and mechanism ofanthocyanidin synthase from Arabidopsis thalianardquo Structurevol 10 no 1 pp 93ndash103 2002

[26] C S Chua D Biermann K S Goo and T-S Sim ldquoElucidationof active site residues of Arabidopsis thaliana flavonol synthaseprovides a molecular platform for engineering flavonolsrdquo Phy-tochemistry vol 69 no 1 pp 66ndash75 2008

[27] K Arnold L Bordoli J Kopp and T Schwede ldquoThe SWISS-MODEL workspace a web-based environment for proteinstructure homology modellingrdquo Bioinformatics vol 22 no 2pp 195ndash201 2006

[28] T Moriguch M Kita K Ogawa Y Tomono T Endo and MOmura ldquoFlavonol synthase gene expression during citrus fruitdevelopmentrdquo Physiologia Plantarum vol 114 no 2 pp 251ndash258 2002

[29] JM Calderon-Montano E Burgos-Moron C Perez-Guerreroand M Lopez-Lazaro ldquoA review on the dietary flavonoidkaempferolrdquo Mini-Reviews in Medicinal Chemistry vol 11 no4 pp 298ndash344 2011

[30] X Feng andW Liu ldquoVariation of quercetin content in differenttissues of welsh onion (Allium fistulosum L)rdquoAfrican Journal ofAgricultural Research vol 6 no 26 pp 5675ndash5679 2011

[31] B Halliwell ldquoFree radicals antioxidants and human diseasecuriosity cause or consequencerdquoTheLancet vol 344 no 8924pp 721ndash724 1994

[32] CG FragaV SMartino andG E Ferraro ldquoFlavonoids as anti-oxidants evaluated by in vitro and in situ liver chemilumines-cencerdquo Biochemical Pharmacology vol 36 no 5 pp 717ndash7201987

[33] A K Ratty and N P Das ldquoEffects of flavonoids on nonenzy-matic lipid peroxidation structure-activity relationshiprdquo Bio-chemical Medicine and Metabolic Biology vol 39 no 1 pp 69ndash79 1988

[34] C Lillo U S Lea and P Ruoff ldquoNutrient depletion as a keyfactor for manipulating gene expression and product formationin different branches of the flavonoid pathwayrdquo Plant Cell andEnvironment vol 31 no 5 pp 587ndash601 2008

[35] D Lewis M Bradley S Bloor et al ldquoAltering expression ofthe flavonoid 31015840-hydroxylase gene modified flavonol ratios andpollen germination in transgenicMitchell petunia plantsrdquoFunc-tional Plant Biology vol 33 no 12 pp 1141ndash1152 2006

[36] R Stracke H Ishihara G Huep et al ldquoDifferential regulationof closely related R2R3-MYB transcription factors controlsflavonol accumulation in different parts of the Arabidopsis

thaliana seedlingrdquo Plant Journal vol 50 no 4 pp 660ndash6772007

[37] S Czemmel R Stracke B Weisshaar et al ldquoThe grapevineR2R3-MYB transcription factor VvMYBF1 regulates flavonolsynthesis in developing grape berriesrdquo Plant Physiology vol 151no 3 pp 1513ndash1530 2009

[38] P Gouet E Courcelle D I Stuart and FMetoz ldquoESPript anal-ysis of multiple sequence alignments in PostScriptrdquo Bioinfor-matics vol 15 no 4 pp 305ndash308 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Zoology


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BioinformaticsAdvances in

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Virolog y

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Nucleic AcidsJournal of

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Stem CellsInternational



Enzyme Research



Microbiology


PhenylalaninePALC4H4CL

Naringenin

Chalcone

CHS

CHI

F3H

DihydroquercetinDihydrokaempferol Dihydromyrcetin

FLS FLSFLS

OOH

O

O

O

O

OKaempferol Quercetin Myricetin

Flavonols

OHOH

OH

OH

OHOH

OH

OH OH

OHOH

OH

OHOH

4-coumaroyl-CoA + 3 malonyl-CoA

F3998400H

F39984005998400H F39984005998400H

Figure 1 Flavonol biosynthesis in plants (redrawn from [37]) The red letter and black box indicate the enzyme and compound analyzed inthis study PAL phenylalanine ammonia lyase C4H cinnamate 4-hydroxylase 4CL 4-coumaroyl CoA ligase CHS chalcone synthase CHIchalcone isomerase F3H flavone 3-hydroxylase F31015840H flavonoid 31015840-hydroxylase F3101584051015840H flavonoid 3101584051015840-hydroxylase FLS flavonol synthase

analyzed by real-time PCR and high-performance liquid chr-omatography (HPLC) The cloning and characterization ofSbFLS may provide a foundation to elucidate the mechanismof flavonol synthesis in S baicalensis

2 Materials and Methods

21 Plant Material and Growth Conditions The seeds of Sbaicalensis were sown in May 2012 and then seedlings weretransferred into pots filled with perlite-mixed soil Seedlingswere grown under 16 h light and 8 h dark conditions in thegreenhouse (25∘C and 50 humidity) at ChungnamNationalUniversity (Daejeon Korea) We used at least nine pots forbiological repeats Each organ (flowers stems leaves androots) was collected for two weeks after the first day of flow-ering All samples were frozen in liquid nitrogen upon collec-tion and stored at minus80∘C prior to RNA isolation and HPLC

22 Cloning of the cDNA Encoding Flavonol Synthase A put-ative flavonol synthase was obtained from S baicalensis bynext-generation sequencing (NGS) (Roche 454 FLX+ andIllumina HiSeq 2000) (unpublished data) We obtained1226938 reads from Roche 454 FLX+ and 161417646 readsfrom Illumina HiSeq 2000 The de novo assembly of the 454and Illumina high-quality reads (95 and 75) were resultedmore than 82Mb sequence lengthwith an average contig readlength of 1614 bp in 51188 contigs which is the long lengthcontigs with at least 500 bp Among these contigs a total of39581 contigs were annotated by BLASTX against the publicNCBI nonredundant database In particular putative SbFLS

sequence was obtained by BLASTX for comparison Theprimers for the open reading frame (ORF) were as followsforward 51015840-ATGGAGGTTGGGAGAGTG-31015840 reverse 51015840-TCACTGGGGAAGCTTATTAAGCTTAC-31015840 The TM Cal-culator program (httpbioinfouteeprimer3-040) wasused to compute the PCR annealing temperatures The PCRprogram consisted of denaturation at 94∘C for 1min anneal-ing at 45∘C for 1min and an extension at 72∘C for 1minThirty cycles were preceded by denaturation step at 94∘Cfor 3min followed by a final extension at 72∘C for 10minThe amplified product was purified and cloned into the T-blunt vector (SolGent Daejeon Korea) and sequenced Thefull-length FLS sequence was aligned using BioEdit SequenceAlignment Editor version 509 [17] For putative conserveddomains we used a GenBank conserved domain databasesearch and function analysis service in NCBI

23 Total RNA Extraction and Quantitative Real-Time RT-PCR Total RNA was isolated from S baicalensis organs withanRNeasy PlantMini Kit (Qiagen Valencia CAUSA) First-strand cDNA was synthesized from total RNA (1 120583g) withReverTra Ace-120572- (Toyobo Osaka Japan) and oligo (dT)

20

primer according to the manufacturerrsquos protocol ORF andgene-specific primers were designed using an onlineprogram (httpfrodowimiteduprimers3) The gene-spe-cific primer sets were designed for SbFLS-RT (forward)51015840-ACCCAACGAGGTTCAAGGAC-31015840 and SbFLS-RT(reverse) 51015840-TGGTGGGGTCTCTTCATTCA-31015840 Real-timePCR was performed in a 20 120583L reaction volume with 05120583Meach primer and 2times SYBR Green real-time PCR master mix







02

CnFLSSbFLS

AmFLSStFLS

NtFLSLjFLS

GtFLSRhFLS

CuFLSRcFLS

VvFLS





D224

H278

H222

QUE

E296

F294

2OG

Y207

R288

(a)

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

11111

7085696979

162178161161171

254270253253264

1205721 1205781 1205731 1205722

1205732 1205723 1205724 1205733 1205734 1205782 1205783

1205725 1205726 1205735 1205736 1205737 1205738 1205739

12057310 12057311 12057312 12057314120573131205727 1205784 1205728

(b)






0

50

100

150

200

250


Gen

e exp

ress

ion

relat

ive t

o ac

tin


0

03

06

09

12


Kaempferol

(mg

g dr

y w

t)

(a)

0

03

06

09

12

15


(mg

g dr

y w

t)

Quercetin

(b)

0

2

4

6

8


Myricetin

(mg

g dr

y w

t)

(c)








4 Conclusion


Abbreviations




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







02

CnFLSSbFLS

AmFLSStFLS

NtFLSLjFLS

GtFLSRhFLS

CuFLSRcFLS

VvFLS





D224

H278

H222

QUE

E296

F294

2OG

Y207

R288

(a)

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

11111

7085696979

162178161161171

254270253253264

1205721 1205781 1205731 1205722

1205732 1205723 1205724 1205733 1205734 1205782 1205783

1205725 1205726 1205735 1205736 1205737 1205738 1205739

12057310 12057311 12057312 12057314120573131205727 1205784 1205728

(b)






0

50

100

150

200

250


Gen

e exp

ress

ion

relat

ive t

o ac

tin


0

03

06

09

12


Kaempferol

(mg

g dr

y w

t)

(a)

0

03

06

09

12

15


(mg

g dr

y w

t)

Quercetin

(b)

0

2

4

6

8


Myricetin

(mg

g dr

y w

t)

(c)








4 Conclusion


Abbreviations




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


D224

H278

H222

QUE

E296

F294

2OG

Y207

R288

(a)

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

SbFLSSbFLS

AtANS

StFLS

CuFLSVvFLS

11111

7085696979

162178161161171

254270253253264

1205721 1205781 1205731 1205722

1205732 1205723 1205724 1205733 1205734 1205782 1205783

1205725 1205726 1205735 1205736 1205737 1205738 1205739

12057310 12057311 12057312 12057314120573131205727 1205784 1205728

(b)






0

50

100

150

200

250


Gen

e exp

ress

ion

relat

ive t

o ac

tin


0

03

06

09

12


Kaempferol

(mg

g dr

y w

t)

(a)

0

03

06

09

12

15


(mg

g dr

y w

t)

Quercetin

(b)

0

2

4

6

8


Myricetin

(mg

g dr

y w

t)

(c)








4 Conclusion


Abbreviations




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

250


Gen

e exp

ress

ion

relat

ive t

o ac

tin


0

03

06

09

12


Kaempferol

(mg

g dr

y w

t)

(a)

0

03

06

09

12

15


(mg

g dr

y w

t)

Quercetin

(b)

0

2

4

6

8


Myricetin

(mg

g dr

y w

t)

(c)








4 Conclusion


Abbreviations




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion


Abbreviations




Acknowledgment


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article cloning and characterization of a...

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