Enhancement of glucosinolate production in watercress (Nasturtium

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Biotechnology and Biological Transformations

Enhancement of glucosinolate production in watercress (Nasturtium officinale) hairy roots by overexpressing cabbage transcription factors Do Manh Cuong, Chang Ha Park, Sun Ju Bong, Nam Su Kim, Jae Kwang Kim, and Sang Un Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00440 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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Journal of Agricultural and Food Chemistry

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Enhancement of glucosinolate production in watercress (Nasturtium officinale) hairy roots

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by overexpressing cabbage transcription factors

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Do Manh Cuong,†,§ Chang Ha Park,†,§ Sun Ju Bong,† Nam Su Kim,† Jae Kwang Kim,*, ‡ Sang

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Un Park*,†

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†

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Daejeon, 34134, Korea

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‡ Division

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Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu,

of Life Sciences and Bio-Resource and Environmental Center, Incheon National

University, Yeonsu-gu, Incheon 22012, Korea

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§

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*To

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J.K. Kim. Division of Life Sciences and Bio-Resource and Environmental Center, Incheon National University, Yeonsu-gu, Incheon 22012, Korea. Phone: +82-32-835-8241. Fax: +82-32835-0763. E-mail: [email protected]

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S.U. Park. Department of Crop Science, Chungnam National University, 99 Daehak-ro,

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Yuseong-gu, Daejeon 34134, Korea. Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail:

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[email protected]

D.M. Cuong and C.H. Park contributed equally to this work. whom correspondence should be addressed

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1 ACS Paragon Plus Environment


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ABSTRACT

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Glucosinolates are secondary metabolites that play important roles in plant defense and human

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health, as their production in plants is enhanced by overexpressing transcription factors. Here,

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four cabbage transcription factors (IQD1-1, IQD1-2, MYB29-1, and MYB29-2) affecting genes

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in both aliphatic and indolic glucosinolates biosynthetic pathways and increasing glucosinolates

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accumulation were overexpressed in watercress. Five IQD1-1, six IQD1-2, five MYB29-1, six

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MYB29-2, and one GUS hairy root lines were created. The expression of all genes involved in

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glucosinolates biosynthesis was higher in transgenic lines than in the GUS hairy root line, in

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agreement with total glucosinolates contents, determined by high-performance liquid

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chromatography. In transgenic IQD1-1(1), IQD1-2(4), MYB29-1(2), and MYB29-2(1) hairy root

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lines total glucosinolates were 3.39-, 3.04-, 2.58-, and 4.69-fold higher than those in the GUS

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hairy root lines, respectively. These results suggest a central regulatory function for IQD1-1,

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IQD1-2, MYB29-1, and MYB29-2 transcription factors in glucosinolates biosynthesis in

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watercress hairy roots.

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KEY WORDS: Watercress, agrobacterium rhizogenes, glucosinolate, hairy root, transcription

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factor

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INTRODUCTION

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Glucosinolates (GSLs) are sulfur-rich secondary metabolites that play important roles in plant

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defense against herbivores and microbes1 and have various biological activities related to human

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health.2-5 These metabolites are found almost exclusively within the order Brassicales including

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watercress (N. officinale),6 rapeseed (B. napus),7 cabbage (B. oleracea),8 Chinese cabbage (B.

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rapa),1 and the model plant A. thaliana.9 In plants, roughly 200 different GSL are accumulation

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occur naturally. According to their amino acid precursor, GSLs are divided into three groups:

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aliphatic GSLs, derived from methionine, isoleucine, leucine, and valine; aromatic GSLs,

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derived from phenylalanine or tyrosine; and indolic GSLs, derived from tryptophan.9 To date,

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almost all genes involved in the GSL biosynthetic pathway have been identified10,11 and are

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known to be positively regulated by several transcription factors (TFs)11 including IQD1-1,

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Dof1.1, MYB28, MYB34, MYB29, MYB51, MYB76, and MYB122.12 MYB28, MYB29, and

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MYB76 control the biosynthesis of aliphatic GSLs,13,14 but MYB34, MYB51, and MYB122

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control the biosynthesis of indolic GSLs,15 and Dof1.1 and IQD1-1 control the biosynthesis of

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both aliphatic and indolic GSLs.16,17 The MYB29 TF, which controls the biosynthesis of high

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aliphatic GSLs, was shown to specifically trans-activate genes of the aliphatic GSL biosynthetic

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pathway (Figure 1), i.e. MAM1-3, CYP79F1, CYP83A1, SUR1, SOT17, and SOT18.13,14,18

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Yimeng Li et al.19 showed that expression levels of BCAT4 were affected by MYB29 gene

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manipulation. The TF IQD1 (IQ67 Domain1) is a founding member of the IQD family that

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affects the expression of multiple genes involved in GSL metabolism.16 Plant-specific IQD gene

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families have been comprehensively annotated in A. thaliana,20,21 rice (O. sativa),20 tomato (S.

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lycopersicum),22 Chinese cabbage (B. rapa),1,23 soybean (G. max),24,25 and black cottonwood (P.

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trichocarpa)26 genomes. In cabbage (B. oleracea), IQD1-1 (Bo1g144340.1), IQD1-2



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(Bo3g061890.1), MYB29-1 (Bo9g175680.1), and MYB29-2 (Bo3g004500.1) TF genes were

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identified and published on http://plants.ensembl.org/Brassica_oleracea/; however, their

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functions have not been studied so far.

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Watercress is an aquatic herb belonging to family Cruciferae (mustard family) rich in

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secondary metabolites such as GSLs, carotenoids, and chlorophyll, and with potential

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antioxidant,27,28 anti-genotoxic, anti-proliferative, and anti-metastatic29 activities. In particular,

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watercress contains high amounts of 2-phenylethyl glucosinolate (gluconasturtiin) and 2-

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phenylethyl isothiocyanate, which participate in tumor growth suppression.30 Several studies

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have recently identified GSLs in watercress hairy roots. Wielanek et al.31 identified

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glucotropaeolin and gluconasturtiin (7.03 and 24.30 mg g-1 dry weight, respectively) in

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watercress hairy roots using high-performance liquid chromatography (HPLC) analysis. Il Park

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et al.6 reported that the levels of indolic GSLs, including glucobrassicin and 4-

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methoxyglucobrassicin, as well as that of the aromatic glucosinolate gluconasturtiin, were

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identical or higher in transformed than in wild type roots of watercress. However, the effects of

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TFs on the accumulation of GSLs in watercress hairy roots have not been examined so far.

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Hairy roots, which are induced by A. rhizogenes R1000 at or near the site of infection of

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a plant, with phenotypic and biochemical stability, al so fast growth rate, they are considered as

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alternative material for the production of secondary metabolites. Given the richness of GSL

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compounds in watercress and the good characteristics of hairy roots, watercress hairy root are

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probably a good model for large-scale production of GSL. In the present study, we enhanced

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GSL production in watercress hairy roots by overexpressing the cabbage TFs IQD1-1, IQD1-2,

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MYB29-1, and MYB29-2, which affect genes in both indolic and aliphatic GSL pathways, as

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well as GSL accumulation. These results will be a valuable resource for further research on 4 ACS Paragon Plus Environment

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watercress bio-engineering and will provide basic information to increase GSL production in

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watercress.

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MATERIALS AND METHODS

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Plasmid Construction. Plasmid construction followed the method of Pham Anh Tuan et

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al..32 A polymerase chain reaction (PCR) was used to generate the coding regions of GUS

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(Accession numbers: AJ298139.1) and IQD1-1, IQD1-2, MYB29-1, and MYB29-2, based on their

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full-length sequences reported in http://plants.ensembl.org/Brassica_oleracea/ with accession

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numbers Bo1g144340.1, Bo3g061890.1, Bo9g175680.1, Bo3g004500.1, respectively. A BP

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Clonase II (Invitrogen, Carlsbad, CA, USA) kit was used to insert these genes into the

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pDONR221 vector (Invitrogen), which was subsequently transformed into TOP10 Escherichia

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coli cells (Invitrogen) by the heat-shock method (42°C for 30 s). Kanamycin (50 mg/L) and

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sequencing methods were used for selecting and confirming gene inserts in the recombinant

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plasmids. Briefly, vectors pDONR221-GUS, pDONR221-IQD1-1, pDONR221-IQD1-2,

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pDONR221-MYB29-1, and pDONR221-MYB29-2 were excised and recombined into a

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pK7FWG2 (http://www.psb.ugent.be/gateway/) overexpression vector using LR Clonase

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(Invitrogen). The resulting constructs, named pK7FWG2-GUS, pK7FWG2-IQD1-1, pK7FWG2-

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IQD1-2, pK7FWG2-MYB29-1, and pK7FWG2-MYB29-2, respectively, consisted of a

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cauliflower mosaic virus (CaMV) 35S promoter, neomycin phosphotransferase (NPTII) gene,

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and GUS, IQD1-1, IQD1-2, MYB29-1, or MYB29-2 sequences, respectively. The electroporation

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method was use to transfer constructs into A. rhizogenes strain R1000, then grown on Luria-

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Bertani medium consists of kanamycin (50 mg/L) at 28 °C and sequenced.



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Plant Material. Watercress (N. officinale) seeds were obtained from Asia Seed Co., Ltd

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(Seoul, Korea). After surface-sterilization with 70% (v/v) ethanol for 30 s, seeds preserved in

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4.5% (v/v) sodium hypochlorite solution for 10 min were rinsed five times in sterilized water,

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germinated on half-strength Murashige and Skoog (1/2 MS) medium33 with 0.8% agar (pH 5.7),

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and incubated at 25°C under 16 h light/8 h dark conditions.

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Induction of Hairy Roots. Leaves of 2-week-old watercress seedlings were exposed to A.

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rhizogenes strain R1000 harboring pK7FWG2-GUS, pK7FWG2-IQD1-1, pK7FWG2-IQD1-2,

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pK7FWG2-MYB29-1, or pK7FWG2-MYB29-2 constructs and cultured on Luria-Bertani (LB)

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medium (sodium chloride 10g/l, trypton 10g/l, yeast extract 5g/l, bactoMT agar 15g/l). After for

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10 min exposure, using sterile filter paper to blotted dry, then watercress leaves were transferred

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to agar-solidified MS medium (pH 5.7), and put in cultivation chamber for 2 days under dark

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light condition at 25°C. After 2 days, watercress leaves washed with sterile distilled water, put in

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agar-solidified 1/2 MS contained 250 mg/L cefotaxime and grown in cultivation chamber under

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dark light for 3 weeks. Hairy roots produced during this period were isolated and cultured

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independently on agar-solidified 1/2 MS with 250 mg/L cefotaxime. Once fully grown, 100 mg

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of each hairy root were transferred to 1/2 MS liquid medium and grown at 25°C and 16 h light/8

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h dark conditions under continuous shaking (100 rpm). After 4 weeks, hairy roots were harvested

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and immediately incubated in liquid nitrogen then stored at - 80°C for analysis of gene

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expression or accumulation GSL compounds.

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Genomic DNA Isolation and PCR Analysis. Total DNAs were isolated from different

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hairy root lines using the Plant Genomic DNA Mini Kit (Geneaid Biotech Ltd., New Taipei City,

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Taiwan), and their quality and concentration were determined by electrophoresis using a 1.2%

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agarose gel and the NanoVue™ Plus spectrophotometer (GE Healthcare UK Limited,


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Buckinghamshire, UK), respectively. To determine if hairy root transformation was successful,

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each hairy root was examined by PCR with primers for Rol and NPTII genes (Table 1). The PCR

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profile used for amplifications was: 95°C for 5 min; 35 cycles of 95°C for 10 s; annealing

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temperature of each gene for 30 s (Table 1), and 72°C for 1 min; final extension at 72°C for 10

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min. After amplification, 20 µL of amplified PCR products were examined by electrophoresis on

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1.2% agarose gels.

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RNA Isolation and cDNA Synthesis. Total RNAs of different hairy root lines were

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isolated using the Easy BLUE Total RNA Kit (iNtRON, Seongnam, Korea). The quality and

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concentration of each RNA were determined by electrophoresis using a 1.2% agarose gel and the

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NanoVue™ Plus spectrophotometer, respectively. The ReverTra Ace-kit (Toyobo Co. Ltd.,

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Osaka, Japan) was used for cDNA synthesis based on 1 μg high-quality total RNA. The cDNA

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samples obtained were diluted 20-fold as template for quantitative real-time PCR (qRT-PCR).

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Quantitative Real-time PCR Analysis. Based on the sequences of the cabbage TFs IQD1-

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1 (Bo1g144340.1), IQD1-2 (Bo3g061890.1), MYB29-1 (Bo9g175680.1), and MYB29-2

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(Bo3g004500.1) published in http://plants.ensembl.org/Brassica_oleracea/Transcript/ and on the

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19 GSL gene sequences reported for watercress,30 Primer 3 (http://bioinfo.ut.ee/primer3-

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0.4.0/primer3/)34 were designed qRT-PCR primers in this study (Table 1). The qRT-PCR

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reaction include cDNA (5 µL), primers (2 µL), water(3 µL), 2× SYBR Green buffer (10 µL)

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with amplification conditions were: 95°C (5 min); 40 cycles of 95°C (20 s), 56°C (20 s), 72°C

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(20 s); and 72°C (8 min). The expression of genes was calculated relative to that of elongation

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factor 1-α (EF-1-α; Accession number: GO479260), which was used as the internal reference

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gene. Each reaction was performed in triplicate and analyzed using the Bio-Rad CFX Manager

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2.0 software (Bio-Rad Laboratories, Hercules, CA, USA).



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HPLC Analysis. Extraction and HPLC analysis of the desulfated forms of GSLs were

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performed as previously reported. 1 GSLs were extracted from 100 mg of freeze-dried sample in

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4.5 mL boiling 70% (v/v) methanol for 5 min, and centrifuged (12,000 rpm) in 10 min at 4°C. To

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elute the extracted GSLs in extract solution, a DEAE-Sephadex A-25 ion exchanger (Sigma-

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Aldrich Korea, Seoul, South Korea) was used. The Agilent Technologies 1200 series HPLC

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system (Palo Alto, CA, USA) including ODS-2 column (10 × 2.0 mm i.d., particle size 5 μm)

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and ODS-3 column (150 × 3.0 mm i.d., particle size 3 μm) from GL Sciences, Tokyo, Japan

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were used to identify GSLs in sample with 227 nm of maximum wavelength, 40°C of column

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oven temperature and 0.2 mL/min of flow rate, respectively. The solvent systems employed were

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water (solvent A) and 100% acetonitrile (solvent B). The following gradient program (27 min in

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total) was used: 0% solvent B, 0 min; 0% to 10% increase in solvent B, 0–2 min; 10% solvent B,

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2–7 min; 10% to 31% steadily increase in solvent B, 7–16 min; 31% solvent B, 16–19 min; 31%

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to 0% decrease in solvent B, 19–21 min; and 0% solvent B, 21–27 min. Individual GSLs were

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identified based on their HPLC peak area ratios, reference to a desulfo-sinigrin external standard,

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and response factors (ISO 9167-1, 1992). Measurements were performed in triplicate and

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analyzed using SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

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Statistical Analysis. For real-time PCR or HPLC statistical analysis, measurements were

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performed in triplicate and analyzed using Bio-Rad CFX Manager 2.0 software (Bio-Rad

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Laboratories, Hercules, CA, USA) or SAS 9.2 (SAS Institute Inc., Cary, NC, USA), respectively.

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RESULTS AND DISCUSSION

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Establishment of Transgenic Hairy Root Lines. After confirming the insertion of IQD1-1,

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IQD1-2, MYB29-1, and MYB29-2 genes by PCR, using primers targeting genes NPTII and Rol


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(data not show), the transcription levels of IQD1-1, IQD1-2, MYB29-1, and MYB29-2 of hairy

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roots from different watercress lines were analyzed by qRT-PCR. High levels of IQD1-1, IQD1-

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2, MYB29-1, and MYB29-2 transcripts were found in the respective transgenic hairy root lines,

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but not in the GUS hairy roots line (Figure 2). Maximum transcript expression levels for IQD1-1,

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IQD1-2, MYB29-1, and MYB29-2 were detected in IQD1-1(1), IQD1-2(4), MYB29-1(2), and

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MYB29-2(1) lines, respectively. Overall, transcript levels were ranked in descending order as

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follows: IQD1-1(1)> IQD1-1(3)> IQD1-1(5)> IQD1-1(4)> IQD1-1(2) for IQD1-1; IQD1-2(4)>

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IQD1-2(6)> IQD1-2(5)> IQD1-2(1)> IQD1-2(2)> IQD1-2(3) for IQD1-2; MYB29-1(2)>

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MYB29-1(4)> MYB29-1(1)> MYB29-1(5)> MYB29-1(3) for MYB29-1; and MYB29-2(1)>

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MYB29-2(3)> MYB29-2(6)> MYB29-2(5)> MYB29-2(2)> MYB29-2(4) for MYB29-2. Lines

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IQD1-1(1), IQD1-2(4), MYB29-1(2), and MYB29-2(1) were selected for further analysis.

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Expression of The Genes Involved in The GSL Biosynthesis Pathway. To investigate the

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regulation of GSL biosynthetic genes by IQD1-1, IQD1-2, MYB29-1, and MYB29-2 TFs, the

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expression levels of NoBCAT4, NoMAM1, NoMAM3, NoCYP79F1, NoCYP83A1, NoGSTF11,

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NoGSTF20, NoGGP1, NoSUR1, NoUGT74C1, NoST5b, NoST5c, NoCYP79B2, NoCYP79B3,

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NoCYP83B1, NoGSTF10, NoUGT74B1, NoST5a, NoCYP81F2, and NoIGMT1 were analyzed in

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IQD1-1(1), IQD1-2(4), MYB29-1(2), MYB29-2(1), and GUS hairy root lines (Figure 3). Genes

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involved in aliphatic GSL biosynthesis include NoBCAT4, NoMAM1, NoMAM3, NoCYP79F1,

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NoCYP83A1, NoGSTF11, NoGSTF20, NoGGP1, NoSUR1, NoUGT74C1, NoST5b, and NoST5c

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and these were expressed to a higher extent in IQD1-1(1), IQD1-2(4), MYB29-1(2), and

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MYB29-2(1) hairy root lines than in GUS hairy root line.

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Expression levels of NoCYP79B2, NoCYP83B1, NoGSTF10, NoUGT74B1, NoST5a,

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NoCYP81F2, and NoIGMT1, which are involved in indolic GSL biosynthesis pathway were also 9 ACS Paragon Plus Environment


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expressed to a higher extent in IQD1-1(1) and IQD1-2(4) hairy root lines than in GUS hairy root

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line. Expression levels of NoCYP79B2, NoCYP79B3, NoCYP83B1, and NoST5a in MYB29-1(2)

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and MYB29-2(1) hairy root lines were similar to that in GUS hairy root line (Figure 3), whereas

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expression levels of NoGSTF10, NoCYP81F2, NoUGT74B1, and NoIGMT1 in MYB29-1(2) and

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MYB29-2(1) hairy root lines were not much higher than in GUS hairy root line. Expression

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levels of NoUGT74B1 and NoIGMT1 were higher in MYB29-2(1) than in MYB29-1(2) hairy

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root line. These results suggested that NoGSTF10, NoCYP81F2, NoUGT74B1, and NoIGMT1

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expression was responsible for the small increase in the accumulation of indolic GSLs observed

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in MYB29-1 and MYB29-2 hairy root lines compared to that observed in the GUS hairy root

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line.

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GSL Production in Lines Overexpressing IQD1-1, IQD1-2, MYB29-1, and MYB29-2.

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According to HPLC results, GSL content significantly increased in transgenic hairy root lines

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compared to the GUS hairy root line (Table 2). Specifically, total GSL content in IQD1-1(1),

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IQD1-2(4), MYB29-1(2), and MYB29-2(1) hairy root lines was 17.77, 15.91, 13.50, and 24.59

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µg/g dry weight, and increased by 3.39-, 3.04-, 2.58-, and 4.69-fold compared to that in the GUS

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hairy root line (5.24 µg/g dry weight), respectively. The accumulation of total aliphatic GSLs

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was higher in transgenic hairy root lines than in the GUS hairy root line. In fact, glucoiberin,

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glucocochlearin, glucobrassicanapin, and glucoerucin, all aliphatic GSLs, were not detected in

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the GUS hairy root line but appeared in transgenic hairy root lines. Furthermore, glucoiberin and

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glucocochlearin were detected in all transgenic hairy root lines, while glucobrassicanapin and

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glucoerucin were only found in MYB29-1(2) and MYB29-2(1) hairy root lines. The

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accumulation of total aliphatic GSLs was highest in MYB29-2(1) (2.23 µg/g dry weight),

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followed by IQD1-2(4) (1.81 µg/g dry weight), MYB29-1(2) (1.58 µg/g dry weight), and IQD1-


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1(1) (1.50 µg/g dry weight), and increased by 3.27-, 2.55-, 2.23-, and 2.11-fold compared to that

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in the GUS hairy root line (0.71 µg/g dry weight), respectively. The accumulation of total indolic

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GSLs was highest in IQD1-1(1) (11.59 µg/g dry weight), followed by IQD1-2(4) (8.98 µg/g dry

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weight), MYB29-2(1) (7.04 µg/g dry weight), and MYB29-1(2) (4.74 µg/g dry weight), and

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increased by 2.85-, 2.21-, 1.73-, 1.17-fold compared to that in the GUS hairy root line (4.06 µg/g

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dry weight), respectively. The accumulation of 4-methoxyglucobrassicin in the MYB29-2(1)

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hairy root line was higher than in the GUS hairy root line, in agreement with the transcript levels

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of NoUGT74B1 and NoIGMT1, which were higher in the MYB29-2(1) hairy root line than in the

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GUS hairy root line. Interestingly, the concentration of gluconasturtiin, a main content of

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aromatic GSL, increased by 9.58-, 10.89-, 15.42-, and 32.42-fold in IQD1-1(1), IQD1-2(4),

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MYB29-1(2), and MYB29-2(1) hairy root lines in relation to the GUS hairy root line (Table 2).

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In the present study, GSL production in watercress hairy roots was enhanced by overexpressing

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the cabbage TFs IQD1-1, IQD1-2, MYB29-1, and MYB29-2, all regulating genes in the indolic

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and aliphatic GSL pathway and GSL accumulation. To assess differences in GSL biosynthetic

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pathways between transgenic and GUS hairy root lines, we compared the accumulation of GSLs

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with the expression levels of the genes involved in their biosynthesis in tissues from 3-week-old

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transgenic watercress hairy root lines and from a GUS hairy root line. Both the GSL profile and

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expression levels of the genes involved in their corresponding biosynthetic pathways were

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considerably distinct between transgenic and GUS hairy root lines. The total GSL content was

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considerably higher in the transgenic hairy root lines than in the GUS hairy root line, in

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agreement with the generally higher transcript levels of the genes related to GSL biosynthesis.

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The overexpression of IQD1-1 and IQD1-2 led to an increase in the expression of genes

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associated with GSL biosynthesis and accumulation (including both aliphatic and indolic GSLs),



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similar to that previously reported by Maggie Levy et al. These authors reported that IQD1

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functions as a positive regulator of GSL accumulation, increasing the content of total GSLs up to

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2-fold that observed in wild-type A. thaliana. While MYb29-1 and MYB29-2 overexpression led

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to an increase in aliphatic GSLs in watercress hairy roots, IQD1-1, IQD1-2, MYB29-1, and

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MYB29-2 overexpression stimulated the expression of genes involved in the GSL biosynthetic

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pathway, inducing GSL accumulation. Similarly, Hirai et al. and Gigolashvili et al. 35,36 reported

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that MYB29 probably played a key role in the regulation of aliphatic GSL biosynthetic genes in

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A. thaliana. Moreover, MYB29 overexpression increased the levels of both short- and long-

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chained aliphatic GSLs in plants.13 In fact, the content of the main short-chained aliphatic

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glucosinolate 4-methylsulfinylbutyl was increased about 2- to 4-fold, and that of 3-

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methylsulfinylpropyl and 5-methylsulfinylpentyl GSLs about 2- to 6-fold in relation to control

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plants. The MYB29 TF was shown to specifically trans-activate genes within the aliphatic GSL

262

biosynthetic pathway, namely, MAM1-3, CYP79F1, CYP83A1, SUR1, SOT17, and SOT18.13,14,18

263

Yimeng Li et al.19 showed that expression levels of BCAT4 were also affected by MYB29 gene

264

manipulation.

265

To our knowledge, this is the first study to enhance GSL production in the hairy roots of

266

watercress by overexpressing the cabbage TFs IQD1-1, IQD1-2, MYB29-1, and MYB29-2.

267

Results obtained here might be useful for elucidating GSL biosynthetic pathways in hairy roots

268

or for targeted genetic engineering, as it is clearly shown that GSL production depends on the

269

examined TFs. Transgenic hairy roots produced higher levels of GSLs and generally exhibited

270

higher gene transcript levels than GUS hairy roots, suggesting that MYB29-1 and MYB29-2 TFs

271

have a central regulatory role in the aliphatic GSL pathway while IQD1-1 and IQD1-2 TFs play

272

a key role in both aliphatic and indolic GSL biosynthesis.


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Page 13 of 25


273

 ABBREVIATIONS USED

274

HPLC, high-performance liquid chromatography; GSLs, glucosinolates; TFs, transcription

275

factors; IQD1, IQ67 Domain1; PCR, polymerase chain reaction;

276

GSL; 4BTEY, 3-butenyl GSL; 4MSOB, 4-methylsulfinylbutyl GSL; 3MTP, 3-methylthiopropyl

277

GSL; 4OHB, 4-hydroxybutyl GSL; 3PREY: 3-propenyl GSL; I3M: indolyl-3-methyl GSL;

278

3MSOP: 3-methylsulfinylpropyl GSL.

279

ASSOCIATED CONTENT

280

Supporting Information Available

4MTB, 4-methylthiobutyl

281 282

AUTHOR INFORMATION

283

Corresponding Authors

284

*J.K.

Kim: Phone: +82-32-835-8241. Fax: +82-32-835-0763. E-mail: [email protected]

285

*S.U.

Park: Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail: [email protected]

286

Author Contributions

287

§

288

Conceived and designed the experiments: J.K. Kim and S.U. Park, Performed the experiments

289

and analyzed the data: D.M. Cuong, C.H. Park, S.J. Bong, and N.S. Kim, Wrote the manuscript:

290

D.M. Cuong and S.U. Park. All authors read and approved the final manuscript.

291

Funding

D.M. Cuong and C.H. Park contributed equally to this work.



292

This research was supported by the Bio & Medical Technology Development Program of the

293

National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning

294

(2016M3A9A5919548).

295 296

Notes

297

The authors declare no competing financial interest.

298

REFERENCES

299

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5. Fahey, J. W.; Zhang, Y. S.; Talalay, P., Broccoli sprouts: An exceptionally rich source of

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9. Halkier, B. A.; Gershenzon, J., Biology and biochemistry of glucosinolates. Annu Rev Plant Biol 2006, 57, 303-333. 10. Sonderby, I. E.; Geu-Flores, F.; Halkier, B. A., Biosynthesis of glucosinolates-gene discovery and beyond. Trends Plant Sci 2010, 15, 283-290. 11. Yan, X. F.; Chen, S. X., Regulation of plant glucosinolate metabolism. Planta 2007, 226, 1343-1352. 12. Wang, H.; Wu, J.; Sun, S.; Liu, B.; Cheng, F.; Sun, R.; Wang, X., Glucosinolate biosynthetic genes in Brassica rapa. Gene 2011, 487, 135-142.

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14. Gigolashvili, T.; Yatusevich, R.; Berger, B.; Muller, C.; Flugge, U. I., The R2R3-MYB

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I.; Gershenzon, J.; Strnad, M.; Szopa, J.; Mueller-Roeber, B.; Witt, I., DOF transcription factor

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Arabidopsis. Plant J 2006, 47, 10-24.

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18. Yatusevich, R.; Mugford, S. G.; Matthewman, C.; Gigolashvili, T.; Frerigmann, H.;

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19. Li, Y.; Sawada, Y.; Hirai, A.; Sato, M.; Kuwahara, A.; Yan, X.; Hirai, M. Y., Novel

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23. Kim, J. K.; Kim, Y. S.; Kim, Y.; Uddin, M. R.; Kim, Y. B.; Kim, H. H.; Park, S. Y.; Lee,

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hairy roots of Scutellaria baicalensis and Scutellaria lateriflora. World J Microbiol Biotechnol

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2014, 30, 887-892. 24. Feng, L.; Chen, Z.; Ma, H.; Chen, X.; Li, Y.; Wang, Y.; Xiang, Y., The IQD gene family

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in soybean: structure, phylogeny, evolution and expression. PLoS One 2014, 9, e110896. 25. Staff, P. O., Correction: the IQD gene family in soybean: structure, phylogeny, evolution

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and expression. PLoS One 2015, 10, e0119318.

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26. Ma, H.; Feng, L.; Chen, Z.; Chen, X.; Zhao, H.; Xiang, Y., Genome-wide identification

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and expression analysis of the IQD gene family in Populus trichocarpa. Plant Sci 2014, 229, 96-

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27. Ozen, T., Investigation of antioxidant properties of Nasturtium officinale (watercress) leaf extracts. Acta Pol Pharm 2009, 66, 187-193.

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28. Gill, C. I. R.; Haldar, S.; Boyd, L. A.; Bennett, R.; Whiteford, J.; Butler, M.; Pearson, J.

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29. Boyd, L. A.; McCann, M. J.; Hashim, Y.; Bennett, R. N.; Gill, C. I.; Rowland, I. R.,

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Assessment of the anti-genotoxic, anti-proliferative, and anti-metastatic potential of crude

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watercress extract in human colon cancer cells. Nutr Cancer 2006, 55, 232-241.



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30. Jeon, J.; Bong, S. J.; Park, J. S.; Park, Y. K.; Arasu, M. V.; Al-Dhabi, N. A.; Park, S. U.,

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R. Br.). BMC genomics 2017, 18, 401.

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31. Wielanek, M.; Krolicka, A.; Bergier, K.; Gajewska, E.; Sklodowska, M., Transformation

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of Nasturtium officinale, Barbarea verna and Arabis caucasica for hairy roots and glucosinolate-

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myrosinase system production. Biotechnol Lett 2009, 31, 917-921.

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32. Tuan, P. A.; Kwon do, Y.; Lee, S.; Arasu, M. V.; Al-Dhabi, N. A.; Park, N. I.; Park, S.

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U., Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by

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over-expression of an Arabidopsis thaliana transcription factor AtPAP1. Int J Mol Sci 2014, 15,

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14743-14752.

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33. Murashige., T.; Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473 - 479. 34. Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B. C.; Remm, M.; Rozen, S. G., Primer3--new capabilities and interfaces. Nucleic Acids Res 2012, 40, e115.

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35. Hirai, M. Y.; Sugiyama, K.; Sawada, Y.; Tohge, T.; Obayashi, T.; Suzuki, A.; Araki, R.;

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Sakurai, N.; Suzuki, H.; Aoki, K.; Goda, H.; Nishizawa, O. I.; Shibata, D.; Saito, K., Omics-

394

based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate

395

biosynthesis. Proc Natl Acad Sci U S A 2007, 104, 6478-83.

396

36. Gigolashvili, T.; Yatusevich, R.; Rollwitz, I.; Humphry, M.; Gershenzon, J.; Flugge, U.

397

I., The Plastidic Bile Acid Transporter 5 Is Required for the Biosynthesis of Methionine-Derived

398

Glucosinolates in Arabidopsis thaliana. Plant Cell 2009, 21, 1813-1829.

399 400

 FIGURE CAPTIONS 18 ACS Paragon Plus Environment

Page 18 of 25

Page 19 of 25


401

Figure 1. The aliphatic and indolic glucosinolate (GSL) biosynthetic pathways in plants. Blue

402

and red colors denote genes and GSL compounds measured in the present study using

403

quantitative real time-PCR and high-performance liquid chromatography analysis, respectively.

404

4MTB, 4-methylthiobutyl GSL; 4MSOB, 4-methylsulfinylbutyl GSL; 4BTEY, 3-butenyl GSL;

405

4OHB, 4-hydroxybutyl GSL; 3MTP, 3-methylthiopropyl GSL; 3MSOP: 3-methylsulfinylpropyl

406

GSL; 3PREY: 3-propenyl GSL; I3M: indolyl-3-methyl GSL.

407

Figure 2. Expression levels of the transcription factor IQD1-1, IQD1-2, MYB29-1, and MYB29-2

408

genes in watercress transgenic hairy root lines. The height of each bar and the error bars show

409

the mean and standard error, respectively, of three independent measurements. GUS, IQD1-1(n),

410

IQD1-2(n), MYB29-1(n), and MYB29-2(n) watercress transgenic hairy root lines were obtained

411

from Agrobacterium rhizogenes strain R1000 containing a pK7FWG2 plant binary expression

412

vector carrying GUS, IQD1-1, IQD1-2, MYB29-1, or MYB29-2, respectively (n indicates the line

413

number) Letters a−e indicate significant differences among organs (P < 0.05).

414

Figure 3. Expression levels of the genes involved in the aliphatic and indolic glucosinolate

415

biosynthesis pathways in watercress transgenic hairy root lines. Three replications were used for

416

each sample in the quantitative real-time PCR analysis conducted to obtained the displayed

417

values. A, B, C, D, and E, represent GUS, IQD1-1(1), IQD1-2(4), MYB29-1(2), and MYB29-

418

2(1) watercress transgenic hairy root lines, obtained from Agrobacterium rhizogenes strain

419

R1000 containing a pK7FWG2 plant binary expression vector carrying GUS, IQD1-1, IQD1-2,

420

MYB29-1, and MYB29-2, respectively. Letters a−e indicate significant differences among organs

421

(P < 0.05).

422 423 424

 TABLES

425

Table 1. Primers used in this study Primers

Forward primer sequences (5’ to 3’)

Primers used for PCR NPTII TAGCCAACGCTATGTCCT RolA CATGTTTCAGAATGGAATTA RolB TCACAATGGATCCCAAATTG

Reverse primer sequences (5’ to 3’)

Tm size (0C) (bp)

GGTGCCCTGAATGAACTG

48

500

AGCCACGTGCGTATTAATCC

43

360

TTCAAGTCGGCTTTAGGCTT

59

900



RolC ATGGCTGAAGACGACCTGTGT RolD ATGGCCAAACAACTTTGCGA Primers used for real-time PCR IQD1-1 TCTCCTCCACAGTTTGAGGTCAGA IQD1-2 TCCTCAGTTTGAGGTCAGAGTTGATG MYB29-1 ACAGTGATGAGACACCGAGAAACAATG MYB29-2 AGCAACCGTGATGAGGTACCGAG EF-1-α ATACCAGGCTTGAGCATACC NoBCAT4 CTGGGACTGCTGCAATCGTG NoMAM1 GCCATTGCACGATGCAAACC NoMAM3 CGGCAGGACGGAGAAGGACT NoCYP79F1 GCGGACCGGCCTCATCTT NoCYP83A1 CGGTGATCGGAAACCTCCAC NoGSTF11 GCGGACCAAGGAACGGATCT NoGSTF20 CCGGTCCTGATCCACAATGG NoGGP1 TGGTGTTGCCCGAATCTGCT NoSUR1 GTCCCGGAGCTGGGATTCTC NoUGT74C1 ATGTGGCGTTCGGGACATTG NoST5b TGACGACTCCTCGAACCCTCTC NoST5c CGTTCGTCGAGTACGGTGGTC NoCYP79B2 CCGATCCTCACGGGACTTGA NoCYP79B3 TTTCAGATGGCTCCACAGCCTTA NoCYP83B1 CGCAGACGCAAAGATTGGTG NoGSTF10 TGCGCCTTTATTCGCTTCTTCA NoUGT74B1 CGTTTGTGGGACGTCATTGGT NoST5a TTCACGCCAAAGACCACTTCG NoIGMT1 GATGTCCCAACCGGAGATGC

Page 20 of 25

TTAGCCGATTGCAAACTTGCA

55

514

TTAATGCCCGTGTTCCATCG

57

1035

TGTAGACTGGATGAACATGGCAGCT

56

225

CTGGATAAAGATGGCAGCTGCTTC

56

181

ATCCTTCCATGGAAGCTGAAAGC

56

147

CCTTCCATGGACGCTGATATGATA

56

134

GCCAAAGAGGCCATCAGACAA

56

117

CCACCGTCCATCCCTTCGTA

56

159

CACGGCCATCTCGATCACTTC

56

156

TGGCGAGGACAACATCCTCA

56

173

GAGGTTGTCCGCTTCGATGG

56

177

CCGGTCCGCAAAGTTGACAT

56

182

GCTCCTCGACCAAAGCGATG

56

172

TCTTGTCTACGAAATCAGCCCAGAA

56

151

GCATCCGCAAATTCTTGCTTG

56

181

GTTTGGTCGAGCCAATGCGT

56

152

GCCACAAGTCCACAGCCTTTCT

56

172

TTCGCAACCGACTCCGGTAA

56

165

CTTCGAATTCTGAACGGTTCGC

56

177

TCGGCAGTAAGCAATGGGTTG

56

200

GAGTGGTCTTGACGCGAAGAGTG

56

151

CCCGACCCGAAAGGTAGGAG

56

192

TCGCGATGTACTCAGGATTCCTCT

56

122

TGGGTCCGATCAACGTAGCCT

56

174

TACGTTTGAGGAGTGGGTTCGTG

56

150

CCCATTCTCTGCGTTGTCAGG

56

159

426 427 428

Table 2. Glucosinolate content (µg/g dry weight) in watercress hairy root lines. ND, not

429

detected. Values are means from three independent experiments ± standard deviation Compounds

GUS

IQD1-1(1)

IQD1-2(4)

MYB29-1(2)

Glucoiberin

ND d

0.30 ± 0.04 a

0.17 ± 0.01 c 0.20 ± 0.01 bc 0.24 ± 0.06 b

Glucoalyssin

0.11 ± 0.00 b

0.14 ± 0.00 a

0.13 ± 0.00 a

Gluconapin

0.60 ± 0.02 c

0.92 ± 0.18 b

1.38 ± 0.11 a 0.72 ± 0.02 bc 1.18 ± 0.22 a

Glucocochlearin

ND c

0.14 ± 0.02 b

0.13 ± 0.01 b

0.15 ± 0.00 b

0.36 ± 0.08 a

Glucobrassicanapin

ND c

ND c

ND c

0.10 ± 0.00 b

0.05 ± 0.01 a

0.14 ± 0.01 a

MYB29-2(1) 0.14 ± 0.00 a

Glucoerucin

ND b

ND b

ND b

0.27 ± 0.01 a

0.35 ± 0.09 a

Total aliphatic GSLs

0.71 ± 0.02

1.50 ± 0.24

1.81 ± 0.13

1.58 ± 0.05

2.32 ± 0.46

4-Hydroxyglucobrassicin

0.69 ± 0.06 c

1.43 ± 0.50 a 1.30 ± 0.12 ab 0.91 ± 0.03 bc 0.87 ± 0.13 c

Glucobrassicin

0.14 ± 0.01 d

1.11 ± 0.09 a

0.57 ± 0.04 b


0.26 ± 0.05 c

0.32 ± 0.08 c

Page 21 of 25


4-Methoxyglucobrassicin Neoglucobrassicin

430

3.19 ± 0.08 c

8.88 ± 1.04 a

6.88 ± 1.05 b

3.52 ± 0.12 c

5.76 ± 0.30 c

0.04 ± 0.00 c 0.17 ± 0.08 ab 0.23 ± 0.12 a 0.05 ± 0.01 bc 0.09 ± 0.00 bc

Total indolic GSLs

4.06 ± 0.15

11.59 ± 1.71

8.98 ± 1.33

Gluconasturtiin

0.47 ± 0.04 d

4.68 ± 0.47 c

5.12 ± 0.16 c

Total GSLs

5.24 ± 0.07

17.77 ± 2.18

15.91 ± 0.71

4.74 ± 0.21

7.25 ± 0.61 b 15.24 ± 2.53 a 13.5 ± 0.59

Letters a−d indicate significant differences among organs (P < 0.05).

431


7.04 ± 0.51

24.59 ± 4.05


432

Page 22 of 25

 FIGURE GRAPHICS

433 Trytophan

Methionine

CY P79B2 CY P79B3

BCAT4

2-keto acid

Indole-3-acetaldoxime

M AM 1/ 2 M AM 3

CY P83B1

Chain elongated methinonine CY P79F1 CY P79F2

S-indol-thiohydroximate

CY P83A1

l-aci-nitro-2-indolyl-ethane

GSTF9 GSTF10

Aldoxime

Aci-nitro compound

GSTF9 GSTF10

GSTF11 GSTF20

Thiohydroximate

S-alkyl-thiohydroximate

UGT74B1

GGP1 SUR1

Desulpho-glucosinolate

Thiohydroximate

ST5a

UGT74G1 UGT74C1

I3M (Glucobrassicin)

Desulpho-Glucosinolate ST5b, ST5c

5-Methylthiopentyl (Glucoberteroin)

4MTB (Glucoerucin)

FM OGS-OX 1 FM OGS-OX 5

3MTP (Glucoibervirin)

FM OGS-OX 1 FM OGS-OX 5

4MSOB 5-Methylsulphinylpentyl (Glucoraphanin) (Glucoalyssin) AOP2

4BTEY (Gluconapin)

3MSOP (Glucoiberin)

AOP2

4-Pentenyl (Glucobrassicanapin)

3PREY (Sinigrin)

10HI3M (1-hydroxyindol -3-ylmethyl)

2-Hydroxy-4-Pentenyl (Gluconapoleif erin)

TGGs Aliphatic glucosinolates

4MOI3M (4-methoxy glucobrassicin) Indolic glucosinolates

1MOI3M (Neoglucobrassicin)

PEN2

Aglucone

434 435

4OHI3M (4-Hydroxy glucobrassicin)

IGM T1 IGM T2

GS-OH

4OHB (Progoitrin)

CY P81F1 CY P81F2 CY P81F3

CY P81F4

Figure 1.


Thiocyanate, Nitrile, Erucin Epithionitrile, Isothiocyanate

Page 23 of 25


436 437

Figure 2.



438 439

Figure 3.

440 441 442 443


Page 24 of 25

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444


Table of Contents Graphics

445 446

Enhancement of glucosinolate production in watercress (Nasturtium officinale) hairy roots

447

by overexpressing cabbage transcription factors

448

Do Manh Cuong,†,§ Chang Ha Park,†,§ Sun Ju Bong,† Nam Su Kim,† Jae Kwang Kim,*, ‡ Sang

449

Un Park*,†

450

451 452


Enhancement of glucosinolate production in watercress (Nasturtium

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