Methyl jasmonate increases isoflavone production in soybean cell

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Bioactive Constituents, Metabolites, and Functions

Methyl jasmonate increases isoflavone production in soybean cell cultures by activating structural genes involved in isoflavonoid biosynthesis Yu Jeong Jeong, Chul Han An, Sung-Chul Park, Jang Won Pyun, Ji-Young Lee, Suk Weon Kim, Hyun-Soon Kim, HyeRan Kim, Jae Cheol Jeong, and Cha Young Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00350 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Journal of Agricultural and Food Chemistry

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Methyl jasmonate increases isoflavone production in soybean cell cultures by activating

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structural genes involved in isoflavonoid biosynthesis

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Yu Jeong Jeong†, Chul Han Ahn†, Sung-Chul Park†, Jang Won Pyun†, Ji-Young Lee†, Suk

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Weon Kim†, Hyun-Soon Kim‡, HyeRan Kim‡, Jae Cheol Jeong*,†, and Cha Young Kim*,†

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† Biological

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(KRIBB), Jeongeup 56212, Republic of Korea

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‡Plant

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Resource Center, Korea Research Institute of Bioscience and Biotechnology

Systems Engineering Research Center, Korea Research Institute of Bioscience and

Biotechnology (KRIBB), Daejeon 34141, Republic of Korea

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*

Co-corresponding authors.

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*

To whom correspondence should be addressed (e-mail: [email protected]).

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Dr. Cha Young Kim

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Tel: +82-63-570-5218

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Fax: +82-63-570-5239

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E-mail: [email protected]

22 23 1

ACS Paragon Plus Environment


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ABSTRACT

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Isoflavonoids are a class of biologically active natural products that accumulate in soybean

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(Glycine max L.) seeds during development, play vital roles in plant defense, and act as

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phytoestrogens with important human health benefits. Plant cell suspension cultures represent

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an excellent source of biologically important secondary metabolites. We found that methyl

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jasmonate (MJ) treatment increased isoflavone production in soybean suspension cell

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cultures. To investigate the underlying mechanism, we examined the expression of structural

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genes (CHS6, CHS7, CHI1, IFS1, IFS2, IFMaT, HID) in the isoflavonoid biosynthesis

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pathways in soybean suspension cells under various abiotic stress conditions. MJ treatment

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had the most significant effect on gene expression and increased the production of three

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glycosidic isoflavones (daidzin, malonyl daidzin, and malonyl genistin), with the maximum

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total isoflavone production (~10-fold increase) obtained on day 9 after MJ application. MJ

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treatment significantly increased total phenolic contents and upregulated isoflavonoid

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biosynthesis genes, shedding light on the underlying mechanism.

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Keywords: soybean, cell cultures, isoflavones, isoflavone biosynthesis, elicitation, methyl

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jasmonate

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INTRODUCTION

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Soybean (Glycine max L.) represents a major source of plant isoflavones for human

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consumption.1-3 Isoflavones are a group of diphenolic secondary metabolites produced by

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soybean and other leguminous plants. Isoflavones act as phytoestrogens, which mimic the

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hormone estrogen, with important beneficial effects on human health.4-5 Many studies

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suggest that the consumption of isoflavones yields numerous health-promoting effects,

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including alleviating menopausal symptoms, preventing osteoporosis and cardiovascular

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diseases, and lowering the risk of breast and prostate cancer.3, 6 In addition, many leguminous

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plants produce isoflavones as defense signaling molecules, such as phytoalexins and

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phytoanticipins, thus protecting the plants from environmental stress.7 Three major types of

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isoflavone aglycones are found in soybean seeds: daidzein, genistein, and glycitein. Most

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isoflavones are conjugated with glucose or malonyl-glucose to become 7-O-β-D glycosides

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(daidzin, genistin, glycitin) and 6”-O-malonyl-7-O-β-D glycosides (malonyldaidzin,

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malonylgenistin, malonylglycitin).5,

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branch of the phenylpropanoid pathway

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which is ubiquitous throughout the plant kingdom, synthesizes a variety of phenolic

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compounds in addition to isoflavones, such as lignans, lignins, flavones, flavonols, condensed

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tannins, and anthocyanins.13,18 Isoflavone synthase (IFS) catalyzes the first committed step of

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isoflavone biosynthesis. IFS converts naringenin synthesized by chalcone isomerase (CHI) to

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geninstein to produce daidzein in conjunction with chalcone reductase (CHR). Both genistein

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and daidzein can be further conjugated sequentially with glucosyl and malonyl side chains

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and become sequestered in the vacuole.13-17

8-12

Isoflavones are synthesized by a legume-specific 13-17

(Figure 1). The phenylpropanoid pathway,

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Plant cell suspension cultures represent a promising alternative source for the production

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and supply of secondary metabolites of pharmaceutical, nutraceutical, and industrial

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importance.19 Plant cells have relatively good doubling times and can be grown aseptically in 3



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simple synthetic liquid medium using conventional bioreactors under controlled

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environmental conditions.20 Plant cell suspension cultures are totally devoid of the problems

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associated with the vagaries of weather, pests, soil, and gene flow in the environment.21 In

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addition, growing plant cells in sterile and controlled environments, such as bioreactor

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systems, allows for precise control over cell growth conditions.22 However, the low

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productivity of secondary metabolites is one of the inevitable obstacles limiting further

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commercialization of plant cell cultures. Secondary metabolites often play an important role

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in plant defense responses, which are triggered and activated by elicitors, the signaling

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molecules of plant defense responses.23 Plant hormones such as salicylic acid (SA), jasmonic

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acid (JA), abscisic acid (ABA), and ethylene (ET) are widely used as elicitors, which

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function as key signals for defense gene expression. Thus, a number of strategies have been

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developed to enhance the production of various products in plants or plant cell cultures.

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Abiotic elicitation is one of the most effective methods for enhancing secondary metabolite

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production.

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In this study, we found that MJ elicitation has positive effects on the expression of a set of

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structural genes involved in isoflavonoid biosynthesis, thereby leading to enhanced

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isoflavone production in soybean suspension cell cultures.

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MATERIALS AND METHODS

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Callus induction and cell suspension cultures

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The soybean calli used in this study were kindly provided by Dr. Mo (Bio FD & C, Korea).

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Callus formation was induced from the shoot apical meristem (SAM) of soybean (Glycine

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max L.), and the calli were maintained at 24°C in the dark on Murashige and Skoog (MS)

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medium (pH 5.8) supplemented with 1.0 mg/L 2,4-D and 30 g/L sucrose (MS1D). The 4


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medium was solidified with 2 g/L Gelrite (Duchefa). The induced calli were subcultured

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every 3 weeks and friable and healthy calli were obtained from several subcultures in the

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same medium. Cell suspension cultures were established by transferring homogeneous calli

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to MS1D liquid medium and were subcultured at 2-week intervals. The culture cells (~1 g in

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fresh weight [FW], 10% v/v inoculum) were transferred into 10 ml MS1D liquid medium in

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125 ml flasks and cultured on a rotary shaker at 90 rpm, 24°C in the dark. Cells were

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harvested via filtration through a nylon mesh filter (50 µm), incubated at 65°C for 48 h, and

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weighed to determine the grams of dry weight (g DW).

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Monitoring of cell viability

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Cell viability was monitored by staining the suspension cells with Evans blue (Sigma-

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Aldrich) as previously described Baker et al (1994).24 Cell viability was scored based on the

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uptake of Evans blue by nonviable cells. Briefly, 1 ml aliquots of culture cells were

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transferred to a new tube and the medium was drained off. The cells were incubated in 1 ml

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of 0.1% Evans blue for 10 min. After the cells were washed several times with deionized

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water to remove excess dye (until no more blue dye was eluted from the cells) and

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resuspended in distilled water, the stained cells (dead cells) were detected under a light

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microscope and images were captured with a digital camera (Motic Moticam Pro 205A).

109 110

Elicitation of soybean cell suspension cultures

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Cell suspension cultures were subcultured into 100 ml MS1D liquid medium in 500 ml

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Erlenmeyer flasks and placed on a rotary shaker (90 rpm) at 24°C under dark conditions for 7

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days. Pre-cultured cells (6 ml aliquots) were transferred into each well of a 6-well tissue

114

plate. Twelve hours later, the cells were treated for 12 h with various elicitors, such as

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salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MJ), ethephon (ET), methyl 5



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viologen (MV), UV, chitosan (CHI), and yeast extract (YE). The cells were harvested by

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filtration through a nylon mesh filter (50 µm) for RNA isolation. The elicitation experiments

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were carried out with three replicates.

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RNA extraction and reverse-transcription (RT)-PCR analysis

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We extracted total RNA from cultured cell samples (~0.1 g) using FavorPrep™Tri-RNA

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reagent (Favorgen). To reduce DNA contamination, RNA cleanup was performed with

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RNeasy Plant Mini Kit (Qiagen) with on-column DNase I treatment according to the

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manufacturer’s instructions. One microgram of total RNA was used to synthesize cDNA

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using a RevertAid™ first-strand cDNA synthesis kit with oligo (dT)18 primer (Thermo

126

Scientific). RT-PCR was performed as described by Chu et al. (2013).25 Beta-tubulin (β-

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TUB) gene was used as an internal control of RT-PCR. The primers used in this study are

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shown in Supplementary Table S1.

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Quantification of isoflavones by HPLC

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Cell suspension cultures were subcultured into 500 ml flasks containing 100 ml (jar volume)

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MS1D liquid medium and incubated on a rotary shaker (90 rpm) at 24°C under dark

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conditions for 5 days. The culture cells were aliquoted into sterile 125 ml flasks (20 ml/flask).

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After 12 h, the cells were elicited with 100 µM MJ for the indicated time periods. Isoflavones

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were extracted from 0.1 g DW of cultured cell samples by shaking for 3 h at room

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temperature in 3 ml of 80% (v/v) methanol. HPLC analysis was performed at 35°C on an

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Agilent 1200 system (Agilent Technologies) equipped with a quaternary pump, an in-line

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degasser, an auto-sampler, and a UV detector using a silica C18 reverse-phase column

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(GEMINI 5 µm C18 125A column, 4.6ｘ150 mm; Phenomenex, Torrance CA, USA) with a 6


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linear gradient of a binary solvent system comprising solvent A (0.5% formic acid in water)

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and solvent B (0.5% formic acid in acetonitrile). The gradient was applied linearly from 95:5

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(A:B, v/v) to 0:100 (A:B, v/v) for 45 min at a flow rate of 1 ml/min. The injection volume

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was 5 µl and the detection wavelength was set to 260 nm. The HPLC standards were

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purchased from Chromadex (Irvine CA, USA), except for the malonyldaidzin and

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malonylgenistin standards, which were purchased from Funakoshi (Tokyo, Japan).

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Quantification of total phenolic contents

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Total phenolic contents in methanolic extracts of soybean suspension cells were determined

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according to the Folin–Ciocalteu method described by An et al. (2015).26 In brief, cultured

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cell samples (1 g FW) were extracted by overnight shaking at room temperature in 3 ml of

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80% (v/v) methanol. After centrifugation, 20 µl of the supernatant was diluted to 850 µl with

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water, followed by the addition of 50 µl Folin–Ciocalteu reagent and 100 µl of 20% (w/v)

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Na2CO3. After 20 min incubation at room temperature, the mixture was measured absorbance

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at 725 nm using a UV-visible spectrophotometer. Chlorogenic acid (CGA) was used as a

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standard of total phenolic content. It was calculated as CGA equivalents using the regression

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equation between CGA standards. The results was expressed as µg CGA equivalents /g fresh

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weight (FW).

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Quantification of total flavonoid contents

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Total flavonoid contents in methanolic extracts of soybean suspension cells were quantified

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as described by Sansanelli et al. (2014).27 A suitable volume of methanolic extract was

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diluted to 400 µl with water and combined with 30 µl of 5% (w/v) NaNO2 solution. After 5

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min incubation, 30 µl of 10% (w/v) AlCl3 was added to the mixture. Additional 6 min 7



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incubation, 200 µl of 1 M NaOH was added. Water was added to the sample up to a total

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volume of 1 ml. The resulting solution was mixed well and the absorbance was immediately

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measured at 510 nm on a UV-visible spectrophotometer. Total flavonoid content was

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calculated as catechin (CAT) equivalents by the regression equation between CAT and was

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expressed as µg CAT equivalents /g FW.

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Quantitative RT-PCR (qRT-PCR)

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Gene-specific primers for soybean were designed using Beacon designer™ software

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(Premier Biosoft). The primer sequences used in the qRT-PCR analyses are shown in

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Supplementary Table 2. Each reaction was performed in a 10 µl volume containing 2 µl of

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cDNA (total 10 ng), 1 µM of each forward and reverse primer, and 5 µl of iQ™ SYBR Green

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Supermix reagent (Bio-Rad) on a CFX96 Real-time system (Bio-Rad). The reactions were

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subjected to an initial denaturation of 95°C for 3 min, followed by 40 cycles of 95°C for

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10 sec, primer-specific annealing, and extension at 55°C for 30 sec. Melting curve analysis

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was performed at the end of the PCR at a temperature range of 65–95°C in 0.5°C

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increments/5 sec. Data were analyzed using CFX manager software ver. 3.1 (Bio-Rad).

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Transcript levels were calculated via normalization against the soybean beta-tubulin (β-TUB)

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gene as a reference control.

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RESULTS AND DISCUSSION

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Growth patterns in soybean cell suspension cultures

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Characterizing cell growth in cell cultures is important for optimizing the culture medium and

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for developing a culture strategy to achieve higher biomass and yields. Thus, we monitored

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cell growth in soybean suspension cultures by measuring cell dry weight (DW). In general,

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cell growth in plant cell suspension cultures displays a sigmoidal-shaped growth curve, with a 8


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lag phase, a logarithmic phase or exponential phase, and a stationary phase.28 As shown in

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Figure 2A, soybean cell growth displayed the typical growth pattern observed in plant cell

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suspension cultures. We observed a lag phase during the first 1–5 days, followed by an

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exponential phase until day 17. During the exponential phase, the cells rapidly divided,

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causing a logarithmic increase in cell density, and the culture medium gradually turned

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brownish. As the cells entered the stationary growth phase, the rate of cell division declined

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and some cells appeared senescent. We monitored cell viability in the soybean cell

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suspension cultures by staining the cells using Evans blue dye (Figure 2B). Living cells

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exclude Evans blue, but dead cells and cell debris are stained blue.24 As shown in Figure 2B,

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less Evans blue dye was retained in cells in the exponential phase compared to those in the

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stationary phase, indicating that cell viability decreased at the stationary phase of growth in

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soybean cell suspension cultures.

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Effects of various elicitors on the expression of structural genes involved in flavonoid

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biosynthesis

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Previous studies reported that four genes such as GmIFS1, GmIFS2, GmCHS7 and GmCHS8

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is mainly involved in isoflavonoid biosynthesis in soybean seeds.29-32 These genes were

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shown to be differentially expressed under drought and temperature stress conditions. The

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varied expression of GmIFS1 and GmIFS2 genes was found to be responsible for the changes

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in isoflavone content in soybean seeds under elicitor treatments.29-30, 33-34 Both GmIFS genes

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were shown to be induced by wounding and MJ application to etiolated soybean seedlings.34

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GmIFS2 was down-regulated by drought stress in soybean plants, resulting in a decrease in

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isoflavone content.31 We previously reported that in soybean plants ethephon treatment

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induces the expression of GmCHS7 and GmIFS2 (structural genes involved in the isoflavone

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biosynthetic pathway) and increases the biosynthesis of isoflavonoids including daidzein and 9



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genistein. To investigate the effects of various elicitors on the activation of isoflavonoid

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biosynthesis in soybean suspension cells, we analyzed GmCHS7 and GmIFS2 expression by

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RT-PCR following elicitation of the cells with SA, ABA, MJ, ET, MV, UV, Chi, and YE. As

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shown in Figure 3A, GmCHS7 and GmIFS2 were upregulated at 12 h in response to the

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application of MJ, YE, and ET. Among these, MJ treatment led to the most significant

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increase in GmCHS7 and GmIFS2 expression levels. We therefore analyzed the expression of

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GmCHS7 and GmIFS2 by RT-PCR after treating the cells with MJ at different time points

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(Figure 3B). Both GmCHS7 and GmIFS2 transcript levels began to increase at 3 h after

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treatment with 100 µM MJ, which was continuously maintained until 48 h. Thus, MJ was

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chosen for further study as an effective elicitor that stimulates isoflavonoid biosynthesis in

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soybean suspension cells.

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We previously reported that ethephon treatment induces the expression of GmCHS7 and

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GmIFS2 in soybean plants. However, in soybean suspension cells, the expression of these

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genes was not significantly altered in response to 100 µM ET but the GmCHS7 gene was

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somewhat induced when treated the cells with 500 µM ET. This discrepancy could be due to

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differences in the elicitation efficiency of ethephon or to the tissue specificity of gene

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expression between soybean plants and suspension culture cells.

232 233

Effects of MJ on isoflavonoid accumulation in soybean suspension cells

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Saini et al. (2013) reported the effect of floral application of several abiotic and biotic

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elicitors on isoflavone levels in soybean seeds.35 Treatment of abiotic and biotic elicitors such

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as SA, MJ, Aspergillus niger and Rhizopus oligosporus elevated total isoflavone levels in

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soybean seeds. The isoflavone content in soybean seeds enhanced with increasing MJ

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concentration whereas it declined with increased concentration of SA. We previously 10


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reported that the biosynthesis of isoflavonoids including daidzein and genistein is induced in

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soybean plants under stress conditions.16 This finding, and the observation that exogenous MJ

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treatment induced the expression of GmCHS7 and GmIFS2, prompted us to analyze the

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isoflavonoid contents in MJ-treated soybean suspension cells via HPLC. In this experiment,

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we used six forms of isoflavone as authentic standards, including the aglycone isoflavones

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daidzein (DE) and genistein (GE), the glycosylated isoflavones daidzin (DI) and genistin

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(GI), and the malonylated isoflavones malonyldaidzin (MDI) and malonylgenistin (MGI).

246

The isoflavonoid contents in soybean suspension cells after MJ application were determined

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by HPLC. As shown in Figure 4A, the DI, MDI, and MGI contents as major isoflavonoids

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greatly increased in MJ-treated suspension cells (9 days after elicitation) but not in untreated

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cells. DI level (12.0 mg/ml) was highest, followed by MDI (6.84 mg/ml), MGI (1.36 mg/ml),

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DE (0.52 mg/ml), GI (0.25 mg/ml), and GE (0.02 mg/ml). These indicate that DI and MDI

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were identified as major isoflavonoids in MJ-treated soybean suspension cells. In addition,

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we investigated the total isoflavone contents in MJ-treated cells at different time points (1–9

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days) using HPLC-DAD. Isoflavone levels gradually increased (from 0.608 to 21.027 mg/g

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DW) until 9 days after MJ application (Figure 4B). In particular, these levels greatly

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increased in soybean suspension cells at 1 day (5.253 mg/g DW) after MJ treatment, whereas

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no significant change was detected in untreated cells. The highest total isoflavone level

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(21.027 mg/g DW) was approximately 10-fold higher than that in untreated control cells

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(2.190 mg/g DW). These results suggest that the activation of GmCHS7 and GmIFS2 by MJ

259

treatment induces isoflavonoid biosynthesis, thereby increasing isoflavonoid contents

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(daidzin and malonyl daidzin as major isoflavonoids) in soybean suspension cells.

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Effects of MJ on total phenolic and total flavonoid contents

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We also measured total phenolic and total flavonoid contents in soybean suspension cells 11



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elicited with MJ via spectrophotometric analyses (Figure 5). Specifically, we monitored total

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phenolic contents by the Folin–Ciocalteu colorimetric method. In control suspension cells,

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total phenolic contents increased from 31.4 µg CGA/g FW on day 0 to 65.2 µg CGA/g FW

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on day 9. By contrast, MJ-treated suspension cells exhibited ~1.7-fold higher total phenolic

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contents (110.8 µg CGA/g FW) on day 9 compared to the control (Figure 5A). In particular,

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the total phenolic content increased ~2-fold on 1 day (59.9 µg CGA/g FW) after MJ

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treatment, whereas a 1.2-fold increase was observed in untreated cells. We also measured

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total flavonoid contents in the same samples. Total flavonoid contents gradually increased

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from day 0 to day 9 in both treated and untreated suspension cells (up to 9.3 and 10.9 µg

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CAT/g FW in control and MJ-treated cells, respectively, on day 9). Total flavonoid contents

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were slightly higher in MJ-treated cells than in control cells (Figure 5B). Together, these

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results indicate that MJ elicitation promotes the accumulation of total phenolics and total

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flavonoids in soybean suspension cells. These findings are in agreement with the increase in

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total isoflavone contents found in MJ-treated cell cultures (Figure 4B).

278 279

MJ application stimulates the phenylpropanoid pathway

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To analyze the relationship between isoflavone accumulation and the expression of

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isoflavonoid biosynthetic genes in MJ-treated soybean suspension cells, we performed

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quantitative RT-PCR (qRT-PCR) analysis to examine the expression of the structural genes

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involved in the isoflavonoid biosynthetic pathways, including CHS, CHI, IFS, HID, IF7GT,

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and IF7MaT. We quantified the relative expression levels of these genes in soybean

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suspension cells elicited for 12 h with MJ compared to the control (Figure 6). IFS1 and IFS2

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were upregulated 5.40- and 11.09-fold, respectively, in response to MJ treatment compared to

287

the control. HID was also strongly induced by MJ treatment (111.96-fold), and IF7MaT was

288

slightly upregulated (3.13-fold) by this treatment, but there was no significant difference in 12


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IF7GT expression in MJ-treated cells (1.39-fold) versus the control. Furthermore, CHS6 and

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CHS7 were also highly upregulated in response to MJ application (by 790.02- and 50.60-fold,

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respectively). CHI1 (8.73-fold) was also induced by MJ, but no significant difference was

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detected in CHI2 expression (0.61-fold) compared to the control. Taken together, these results

293

suggest that eliciting soybean suspension cells with MJ induces isoflavonoid biosynthesis via

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the activation of structural genes in both the flavonoid pathway (CHS6, CHS7, CHI1) and the

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isoflavonoid biosynthetic pathway (IFS1, IFS2, IFMaT, HID).

296

In conclusion, we demonstrated the improvement in the isoflavone content in soybean cell

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suspension cultures through the elicitor (MJ)-mediated approach. This was correlated with

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the up-regulation of isoflavone biosynthetic genes during MJ elicitation. Among isoflavones,

299

daidzin (DI) and malonyl daidzin (MDI) accumulated to high levels in soybean cell

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suspension cultures by MJ application. The elicitor-mediated approach could potentially be

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used to increase isoflavone content in soybean plants and cell cultures. In addition to soybean

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seeds, soybean suspension cell cultures can be used as cell factories to produce isoflavones

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by activating isoflavonoid biosynthetic pathway through MJ elicitation. Since soy isoflavones

304

have great opportunities for preventive and therapeutic use for several diseases, they can be

305

used as foods or food supplements for humans and animals. Thus, we are currently

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conducting experiments for optimization of soybean cell suspension cultures to maximize the

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cell growth and isoflavone contents through scale-up cultivation in bioreactors.

308 309

Acknowledgments

310 311

This research was supported by grants from the KRIBB Initiative Program, the Next-

312

Generation BioGreen 21 Program (SSAC, Grant #: PJ01318604), Rural Development

313

Administration, and the Bio & Medical Technology Development Program of the National 13



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Research Foundation funded by the Korean government, Republic of Korea.

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Supporting Information

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Table S1. Primer sequences used for RT-PCR in this study Primer name

Accession no.

Primer sequence

CHS7-For

M98871

5’-CCTTTGTATGAGCTTGTTTGGAC-3’

CHS7-Rev

M98871

5’-CCAAGCAAGTTTGAAAAGTGGTA-3’

IFS2-For

AF195799

5’-TAATGGATATGTGATCCCAGAGG-3’

IFS2-Rev

AF195799

5’-TGCATGGAAGGGCTTATATACTT-3’

β-TUB-For

X60216

5’-CAAATGTGGGATGCTAAGAACAT-3’

β-TUB-Rev

X60216

5’-AACTTACACCACTCGTTCAAAGC-3’

319 320

Primers were designed using Primer3 software (Rozen and Skaletsky, 2000).

321 322 323

Table S2. Primer pairs used for qRT-PCR in this study Gene

Gene name

Accession no.

Primer sequence (forward/reverse)

Product size (bp)

5’-CTCACTTTCCATCTCCTCA-3’ CHS6

CHS7

CHI1

CHI2

IFS1

Chalcone synthase 6

L03352

Chalcone synthase 7

M98871

116 5’-ATCCAAAAGATAGAGTTGTAATCATC-3’ 5’-TCGCCCTTATGTGAAGAGGTAC-3’ 127 5’-CTCAGAGCAGACAACAAGCA-3’ 5’-TGGGACTTACAGTGAAGCA-3’

Chalcone isomerase 1

AY595419

Chalcone isomerase 2

DQ191404

Isoflavone

AF195798

83 5’-TGGAGCCTGGTGGGAAAT-3’ 5’-CCAGGCTCCACTGTTTTCTAC-3’ 149 5’-TCTCCGATCATAGTCTCCAGC-3’ 5’-TAAGACGCCTCACTTACGAC-3’

141 15



synthase 1

IFS2

HID

IF7GT

IF7MaT

Page 16 of 38

5’-GGATCTGTTGGGTCCTCAAAG-3’ 5’-CATAAGACGCCTCACCTATGATAG-3’

Isoflavone synthase 2

AF195799

2Hydroxyisoflavano ne dehydratase

NM 001250299

UDP-glucose: isoflavone 7-Oglucosyl transferase

NM 001248232

Malonyl-CoA: isoflavone 7-Oglucoside-6-Omalonyltransferas e

NM 001250831

Beta-tubulin

X60216

122 5’-GCCTCAACTTGTTTACAGTGGT-3’ 5’-CACTCTTGCCTGCTCTAAGTT-3’ 88 5’-CTCAACGGTGTGGTGGTAG-3’ 5’-CCCCACCATTCACCCAAC-3’ 135 5’-TGGCAAGCGTAACTCAAGG-3’ 5’-CCCTCTCTTCAAACCTCTCAG-3’ 148 5’-TGGTGGCTTGTTATTCCTATCG-3’ 5’-GCTGATGAGTGTATGGTTTTG-3’

β-TUB

150 5’-TTGCCCAGGGAAACG-3’

324 325

Primers were designed using Beacon designer™ software (Premier Biosoft).

326 327 328 329 330 331 332 333 334 335 336 16


Page 17 of 38

337


References

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isoflavone 7-O-glucosyltransferase from Pueraria lobata. Plant Cell Rep. 2014, 33 (7), 1173-

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hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in

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leguminous isoflavone biosynthesis. Plant Physiol. 2005, 137 (3), 882-891.

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Suzuki, H.; Nishino, T.; Nakayama, T. cDNA cloning of a BAHD acyltransferase

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from

soybean

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Phytochemistry 2007, 68 (15), 2035–2042.

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Uddin, Z.; Kim, C. Y.; Park, K. H., Ethylene induced a high accumulation of dietary

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isoflavones and expression of isoflavonoid biosynthetic genes in soybean (Glycine max)

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leaves. J. Agric. Food Chem. 2016, 64 (39), 7315-7324.

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isoflavone synthase gene from Psoralea corylifolia: a medicinal plant. Plant Cell Rep. 2010,

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Shi, H.; Nam, P. K.; Ma, Y., Comprehensive profiling of isoflavones, phytosterols,

Li, J.; Li, Z.; Li, C.; Gou, J.; Zhang, Y., Molecular cloning and characterization of an

Yu, O.; Shi, J.; Hession, A. O.; Maxwell, C. A.; McGonigle, B.; Odell, J. T.,

Akashi, T.; Aoki, T.; Ayabe, S. Molecular and biochemical characterization of 2-

(Glycine

max):

isoflavone

7-O-glucoside-6’’-O-malonyltransferase.

Yuk, H. J.; Song, Y. H.; Curtis-Long, M. J.; Kim, D. W.; Woo, S. G.; Lee, Y. B.;

Misra, P.; Pandey, A.; Tewari, S. K.; Nath, P.; Trivedi, P. K., Characterization of

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Vogt, T., Phenylpropanoid biosynthesis. Mol. Plant 2010, 3 (1), 2-20.

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hairy root cultures of Glycyrrhiza uralensis Fisch by combining the over-expression of

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chalcone isomerase gene with the elicitation treatment. Plant Cell Rep. 2009, 28 (8), 1205-

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1213.

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proteins with plant cell suspension cultures. Biotechnol. Adv. 2011, 29 (3), 278-299.

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G. G. d.; Gomord, V., Production of recombinant proteins in suspension–cultured plant cells.

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In Recombinant Proteins From Plants: Methods and Protocols, Faye, L.; Gomord, V., Eds.

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Humana Press: Totowa, NJ, 2009, pp 145-161.

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commercialization of plant cell culture processes for the synthesis of biomolecules. Plant

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Biotechno. J. 2012, 10 (3), 249-268.

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cell systems. Biotechnol. Appl. Biochem. 2003, 37 (1), 91-102.

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suspension and leaf disc assays using evans blue. Plant Cell, Tissue Organ Cult. 1994, 39 (1),

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7-12.

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H.; Lee, H.-S.; Kwak, S.-S.; Kim, C. Y., Expression of the sweetpotato R2R3-type IbMYB1a

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gene induces anthocyanin accumulation in Arabidopsis. Physiol. Plant 2013, 148 (2), 189-

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199.

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Kwak, S.-S.; Kim, C. Y., Heterologous expression of IbMYB1a by different promoters

Xu, J.; Ge, X.; Dolan, M. C., Towards high-yield production of pharmaceutical

Plasson, C.; Michel, R.; Lienard, D.; Saint-Jore-Dupas, C.; Sourrouille, C.; March,

Wilson, S. A.; Roberts, S. C., Recent advances towards development and

Radman, R.; Saez, T.; Bucke, C.; Keshavarz, T., Elicitation of plants and microbial

Jacyn Baker, C.; Mock, N. M., An improved method for monitoring cell death in cell

Chu, H.; Jeong, J. C.; Kim, W.-J.; Chung, D. M.; Jeon, H. K.; Ahn, Y. O.; Kim, S.

An, C. H.; Lee, K.-W.; Lee, S.-H.; Jeong, Y. J.; Woo, S. G.; Chun, H.; Park, Y.-I.;

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exhibits different patterns of anthocyanin accumulation in tobacco. Plant Physiol. Biochem.

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2015, 89, 1-10.

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and glycosylated isoflavones in in vitro soybean (Glycine max L.) hypocotyl cell suspensions

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and comparison with industrial seed extracts. Plant Cell, Tissue and Organ Cult. 2014, 119

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(2), 301-311.

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cryopreservation of plant cell suspension cultures. Nat. Protoc. 2011, 6 (6), 715-742.

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reveals a critical role of CHS7 and CHS8 genes for isoflavonoid synthesis in soybean seeds.

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Plant Physiol. 2007, 143 (1), 326-338.

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with isoflavone concentrations in soybean seeds. Plant Sci. 2008, 175 (4), 505-512.

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R.; Yu, O.; Nguyen, H. T.; Sleper, D. A., Differential expression of isoflavone biosynthetic

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genes in soybean during water deficits. Plant Cell Physiol. 2010, 51 (6), 936-948.

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stress on soybean isoflavone concentration and expression of key genes involved in

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isoflavone synthesis. J. Agric. Food Chem. 2012, 60 (51), 12421-12427.

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isoflavone synthase genes respond differentially to nodulation and defense signals in

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transgenic soybean roots. Plant Mol. Biol. 2004, 54 (5), 623-639.

432

34.

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I.; Kim, J. K., Polymorphism and expression of isoflavone synthase genes from soybean

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cultivars. Mol. Cells 2005, 19 (1), 67-73.

Sansanelli, S.; Zanichelli, D.; Filippini, A.; Ferri, M.; Tassoni, A., Production of free

Mustafa, N. R.; de Winter, W.; van Iren, F.; Verpoorte, R., Initiation, growth and

Dhaubhadel, S.; Gijzen, M.; Moy, P.; Farhangkhoee, M., Transcriptome analysis

Cheng, H.; Yu, O.; Yu, D., Polymorphisms of IFS1 and IFS2 gene are associated

Gutierrez-Gonzalez, J. J.; Guttikonda, S. K.; Tran, L.-S. P.; Aldrich, D. L.; Zhong,

Chennupati, P.; Seguin, P.; Chamoun, R.; Jabaji, S., Effects of high-temperature

Subramanian, S.; Hu, X.; Lu, G.; Odelland, J. T.; Yu, O., The promoters of two

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Saini Ramesh, K.; Akithadevi Muthu, K.; Giridhar, P.; Ravishankar Gokare, A.,

436

Augmentation of major isoflavones in Glycine max L. through the elicitor-mediated

437

approach. In Acta Bot. Croat., 2013, Vol. 72, p 311.

21



438

Page 22 of 38

Figure captions

439 440

Figure 1. Proposed isoflavonoid biosynthetic pathway in soybean. The sequential actions of

441

CHS, CHR, CHI, IFS, HID, IF7GT, and IF7MaT result in the conversion of phenylalanine to

442

isoflavones. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-

443

coumarate-CoA-ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone

444

isomerase; IFS, isoflavone synthase; HID, 2-hydroxyisoflavanone dehydratase; IF7GT, UDP-

445

glucose: isoflavone 7-O-glucosyltransferase; IF7MaT, malonyl-CoA: isoflavone 7-O-

446

glucoside-6-O-malonyltransferase.

447 448 449

Figure 2. The growth of soybean suspension culture cells. (A) Growth curve of cultured cells

450

at different time points. Three replications were performed for each sample. (B) Evans blue

451

staining for cell viability at different stages, including the exponential phase (left panels) and

452

stationary growth phase (right panels). Scale bar indicates 50 µm.

453 454 455

Figure 3. Effect of various elicitors on the expression of isoflavone biosynthetic genes in

456

soybean suspension cell cultures. (A) Cells were harvested at 12 h after elicitation for RT-

457

PCR analysis. The elicitors used are as follows: ET, ethephon; MV, methyl viologen; Chi,

458

chitosan; YE, yeast extract. (B) Time-course of gene expression after MJ and YE treatment.

459

The soybean isoflavone biosynthetic genes used for RT-PCR are as follows: CHS7, chalcone

460

synthase 7; IFS2, isoflavone synthase 2. Soybean β-tubulin was used as a quantitative control.

461

22


Page 23 of 38


462 463

Figure 4. Effects of MJ on isoflavone production in soybean suspension cells. (A) HPLC

464

analysis was performed using a C18 reverse-phase column at 260 nm. Chromatogram STD

465

represents the authentic standards DI (daidzin), GI (genistin), MDI (malonyl daidzin), MGI

466

(malonyl genistin), DE (daidzein), and GE (genistein) with retention times of 5.928, 9.248,

467

9.712, 15.018, 17.309, and 21.755 min, respectively. Chromatogram MJ indicates the

468

isoflavone production in the elicited cells at 9 days after elicitation with 100 µM MJ. (B)

469

Time-course of isoflavone production in soybean suspension cells elicited with MJ. The cells

470

were pre-cultured for 5 days and harvested at the indicated time points after elicitation with

471

100 µM MJ. Samples were extracted with methanol and subjected to HPLC analysis. Total

472

isoflavone contents were calculated as the sum of the contents of the six forms of isoflavone

473

(DI, GI, MDI, MGI, DE, and GE).

474 475 476

Figure 5. Total phenolic contents (A) and total flavonoid contents (B) in soybean suspension

477

cells elicited with MJ. The analyses were performed by spectrophotometric quantification.

478

Total phenolic contents are expressed as µg of chlorogenic acid (CGA) equivalent per g FW

479

(µg CGA/g FW); total flavonoid contents are expressed as µg of catechin (CAT) equivalent

480

per g FW (µg CAT/g FW).

481 482 483

Figure 6. Relative expression of isoflavone biosynthetic genes in MJ-treated soybean

484

suspension cells. CHS, CHI, IFS, HID, IF7GT, and IF7MaT mRNA levels were analyzed by

485

qRT-PCR using soybean suspension cells elicited with MJ for 12 h. Data were normalized 23



Page 24 of 38

486

against the expression of the housekeeping gene beta-tubulin (β-TUB). To determine the

487

relative fold differences for each gene in each experiment, the Ct values of the genes were

488

normalized to the Ct value for tubulin, and relative expression was calculated relative to a

489

calibrator using the formula 2-∆∆Ct. All values shown are mean ± SE.

490

24


Page 25 of 38

491


Figures

492

493 494 495 496

Figure 1

497

25



Page 26 of 38

498 499 500

Figure 2

501

26


Page 27 of 38


502 503 504

Figure 3

505

27



Page 28 of 38

506 507 508

Figure 4

509

28


Page 29 of 38


510 511 512

Figure 5

513

29



Page 30 of 38

514 515 516

Figure 6

517

30


Page 31 of 38

518


Graphic Abstract

519

520

31



Figure 1. Proposed isoflavonoid biosynthetic pathway in soybean. The sequential actions of CHS, CHR, CHI, IFS, HID, IF7GT, and IF7MaT result in the conversion of phenylalanine to isoflavones. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate-CoA-ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; HID, 2-hydroxyisoflavanone dehydratase; IF7GT, UDP-glucose: isoflavone 7-O-glucosyltransferase; IF7MaT, malonyl-CoA: isoflavone 7O-glucoside-6-O-malonyltransferase. 119x219mm (300 x 300 DPI)


Page 32 of 38

Page 33 of 38


Figure 2. The growth of soybean suspension culture cells. (A) Growth curve of cultured cells at different time points. Three replications were performed for each sample. (B) Evans blue staining for cell viability at different stages, including the exponential phase (left panels) and stationary growth phase (right panels). Scale bar indicates 50 µm. 80x129mm (300 x 300 DPI)



Figure 3. Effect of various elicitors on the expression of isoflavone biosynthetic genes in soybean suspension cell cultures. (A) Cells were harvested at 12 h after elicitation for RT-PCR analysis. The elicitors used are as follows: ET, ethephon; MV, methyl viologen; Chi, chitosan; YE, yeast extract. (B) Time-course of gene expression after MJ and YE treatment. The soybean isoflavone biosynthetic genes used for RT-PCR are as follows: CHS7, chalcone synthase 7; IFS2, isoflavone synthase 2. Soybean β-tubulin was used as a quantitative control. 80x80mm (300 x 300 DPI)


Page 34 of 38

Page 35 of 38


Figure 4. Effects of MJ on isoflavone production in soybean suspension cells. (A) HPLC analysis was performed using a C18 reverse-phase column at 260 nm. Chromatogram STD represents the authentic standards DI (daidzin), GI (genistin), MDI (malonyl daidzin), MGI (malonyl genistin), DE (daidzein), and GE (genistein) with retention times of 5.928, 9.248, 9.712, 15.018, 17.309, and 21.755 min, respectively. Chromatogram MJ indicates the isoflavone production in the elicited cells at 9 days after elicitation with 100 µM MJ. (B) Time-course of isoflavone production in soybean suspension cells elicited with MJ. The cells were pre-cultured for 5 days and harvested at the indicated time points after elicitation with 100 µM MJ. Samples were extracted with methanol and subjected to HPLC analysis. Total isoflavone contents were calculated as the sum of the contents of the six forms of isoflavone (DI, GI, MDI, MGI, DE, and GE). 80x119mm (300 x 300 DPI)



Figure 5. Total phenolic contents (A) and total flavonoid contents (B) in soybean suspension cells elicited with MJ. The analyses were performed by spectrophotometric quantification. Total phenolic contents are expressed as µg of chlorogenic acid (CGA) equivalent per g FW (µg CGA/g FW); total flavonoid contents are expressed as µg of catechin (CAT) equivalent per g FW (µg CAT/g FW). 80x114mm (300 x 300 DPI)


Page 36 of 38

Page 37 of 38


Figure 6. Relative expression of isoflavone biosynthetic genes in MJ-treated soybean suspension cells. CHS, CHI, IFS, HID, IF7GT, and IF7MaT mRNA levels were analyzed by qRT-PCR using soybean suspension cells elicited with MJ for 12 h. Data were normalized against the expression of the housekeeping gene betatubulin (β-TUB). To determine the relative fold differences for each gene in each experiment, the Ct values of the genes were normalized to the Ct value for tubulin, and relative expression was calculated relative to a calibrator using the formula 2-∆∆Ct. All values shown are mean ± SE. 80x119mm (300 x 300 DPI)



58x44mm (300 x 300 DPI)


Page 38 of 38

Methyl jasmonate increases isoflavone production in soybean cell

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