Characterization of Stilbene Synthase Genes in mulberry (Morus

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Characterization of Stilbene Synthase Genes in mulberry (Morus atropurpurea) and Metabolic Engineering for the Production of Resveratrol in Escherichia coli Chuanhong Wang, Shuang Zhi, Changying Liu, Fengxiang Xu, Ai Chun Zhao, Xiling Wang, Yanhong Ren, Zhengang Li, and Maode Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05212 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Agricultural and Food Chemistry

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Characterization of Stilbene Synthase Genes in mulberry (Morus atropurpurea)

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and Metabolic Engineering for the Production of Resveratrol in Escherichia coli

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Chuanhong Wang1, Shuang Zhi1, Changying Liu1, Fengxiang Xu1, Aichun Zhao1,

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Xiling Wang1, Yanhong Ren1, Zhengang Li2, Maode Yu1*

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District, Chongqing 400716, China

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2

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Agricultural Sciences, Mengzi, Yunnan 661100, China

College of Biotechnology, Southwest University, No.2 Tiansheng Road, BeiBei

The Sericultural and Apicultural Research Institute, Yunnan Academy of

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* Corresponding author: Maode Yu

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E-mail: [email protected]

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Tel:+8618723079257

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Fax: +86-023-68250191

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ACS Paragon Plus Environment


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Abstract

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Stilbenes have been recognized for their beneficial physiological effects on

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human health. Stilbene synthase (STS) is the key enzyme of resveratrol biosynthesis

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and has been studied in numerous plants. Here, four MaSTS genes were isolated and

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identified in mulberry (Morus atropurpurea Roxb.). The expression levels of MaSTS

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genes and the accumulation of trans-resveratrol,

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trans-mulberroside A were investigated in different plant organs. A novel

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co-expression system that harbored 4-coumarate:CoA ligase gene (Ma4CL) and

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MaSTS was established. Stress tests suggested that MaSTS genes participates in

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responses to salicylic acid, abscisic acid, wounding and NaCl stresses. Additionally,

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over-expressed MaSTS in transgenic tobacco elevated the trans-resveratrol level and

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increased tolerance to drought and salinity stresses. These results revealed the major

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MaSTS gene and we evaluated its function in mulberry, laying the foundation for

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future research on stilbene metabolic pathways in mulberry.

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Keywords: mulberry; stilbene synthase; stilbenes; resveratrol; co-expression

trans-oxyresveratrol and

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Introduction

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Stilbenes are non-flavonoid polyphenols, derived from the phenylpropanoid

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pathway, that function as phytoalexins in plants to resist various biotic and abiotic

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stresses, like UV radiation, bacteria, fungi and herbivores.1-4 To date, stilbenes have

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been identified in more than 70 unrelated plant species, including grape, apple, peanut,

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pine, rhubarb and sorghum.1,3,5,6 In recent decades, many studies on the physiological

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functions of stilbenes in vivo/vitro indicated that stilbenes have significant

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health-promoting effects on the body, such as the prevention of cancer, heart disease

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and neurodegenerative diseases, and inhibiting α-glucosidase activities and tyrosinase

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gene expression.7−10

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Stilbene synthase (STS) is the pivotal enzyme in the synthesis of stilbenes. It

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occurs in a limited number of plant species and utilizes a tetraketide intermediate that

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condenses with three malonyl-CoA and one 4-coumaroyl-CoA molecules (Figure 1).

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Interestingly, as another member of polyketide synthase III, chalcone synthase (CHS)

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utilizes the same starter phenylpropanoid-CoA esters as STS (Figure 1). Moreover,

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because CHS and STS have a high degree of similarity at the amino acid level,

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numerous plant STS sequences are annotated as CHS in different public databases

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based on sequence homology.11 However, these genes may in fact have other

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metabolic roles, such as stilbene-forming activities.11,12

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Mulberry leaves are widely known for their role in the silk production in Asian

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countries. Human utilization of the mulberry–silkworm interaction began at least

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5,000 years ago and greatly influenced human civilization.13 Different parts of the 3



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mulberry have been extensively investigated in recent years, and the plant is a good

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source of distinct natural products that are able to positively impact human health,

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including antioxidative, antihyperglycemic, hypolipidemic and antiatherogenic effects

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and the inhibition of α-glucosidase activities.8,14 In addition, mulberry is rich in

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resveratrol, oxyresveratrol and mulberroside A.8,15,16 Unfortunately, compared with

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information on the bioactivity of stilbenes in mulberry, there are limited reports

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related to MaSTS. Thus, it is necessary to study the functions of MaSTS genes in

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mulberry.

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To investigate the mechanism of MaSTS in mulberry, we determined the levels of

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the three main stilbenes of ‘Guiyou No. 62’ (Morus atropurpurea Roxb.), and we

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evaluated four MaSTS gene responses to salicylic acid (SA), abscisic acid (ABA),

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wounding and NaCl stresses. Moreover, stilbenes in the fruit of a new cultivated

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variety ‘Jialing No. 40’ (Morus atropurpurea Roxb.) (tetraploid, ‘Zhongsang5801’ ×

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‘Naxi’, hypocotyl chromosome doubling, 2n = 4x = 56) were also investigated.

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Additionally, we established a co-expression method to produce trans-resveratrol in

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Escherichia coli. The tolerance of transgenic tobacco harboring MaSTS to multiple

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abiotic stresses was also evaluated.

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Materials and methods

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Data retrieval and cloning of MaSTS cDNAs

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Similarity searches were performed using the coding region of the Fallopia

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multiflora STS (AGA35552.1), Arachis hypogaea STS (BAA78617.1), and

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Polygonum cuspidatum STS (ACC76753.1) against the Morus Genome Database 4


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(http://morus.swu.edu.cn/morusdb/). The candidate genes were identified using

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BLASTN and SMART (http://smart.embl-heidelberg.de/). The purified PCR products

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were cloned into the pMD19-T simple vector (Takara, Otsu, Japan) and sequenced.

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Plant materials and abiotic stress test

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The mulberry fruit materials were collected from the mulberry cultivar ‘Jialing

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No.40’ at seven different developmental stages (S1–S7), and the leaves, stems bark

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and roots bark were excised from mature plants of ‘Guiyou No. 62’ in the mulberry

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garden of Southwest University. Abiotic stress tests were performed as previously

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described.17 Briefly, 1 week-old mulberry seedlings were used for ABA (50 µM), SA

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(5 mM) and NaCl (50 mg/L) treatments, and 10-week-old mulberry plants were used

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for mechanical wounding treatments. The wounding treatment consisted of creating

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eight wounds along leaf veins using a sterile toothpick. All of the materials were

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immediately frozen in liquid nitrogen and stored at −80°C, and/or freeze-dried, for

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RNA isolation and/or high performance liquid chromatography (HPLC) analysis,

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respectively.

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RNA extraction, cDNA synthesis and quantitative real-time polymerase chain

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reaction (qRT-PCR)

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The total RNA of mulberry fruit was extracted using an RNA Extraction TransZol

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Plant Kit (TransGen Biotech, Beijing, China), and the other tissues’ total RNA were

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extracted using an RNA Extraction Kit (TaKaRa, Dalian, China), The RNA samples

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were treated with DNase I (TaKaRa) to digest genomic DNA, and 2 µg of purified

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RNA was used to synthesize cDNA with Moloney murine leukemia virus reverse 5



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transcriptase (Promega, Madison, WI, USA). Six-fold-diluted cDNA was used in

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RT-PCR and qRT-PCR. The primers were designed using the online tool of the

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GeneScript Company (Nanjing, China) (http://www.genscript.com.cn/index.html)

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(Table S1 and Table S2). The qRT-PCR was performed according to the

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manufacturer’s instructions for SYBR® Premix Ex TaqTM II (TaKaRa) and conducted

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in the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

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To each reaction, 2 µL of diluted cDNA was added, and the MaActin3 gene was used

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as an internal control to normalize the relative expression of target genes. All of the

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data was analyzed using the 2−∆∆Ct method.17,22

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Determination of stilbenes in different tissues

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Different parts of the mulberry were collected and dried in a vacuum freeze drier

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(Thermo Fisher Scientific, Waltham, MA, USA). Each sample was ground in liquid

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nitrogen, 1 mL of 75% methanol was added per 0.1 g of powder, and extracted for 30

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min three times by ultrasonication. The filtrate was evaporated to dryness and

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dissolved in 5 mL of methyl alcohol. Each sample was centrifuged and filtered

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through

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trans-oxyresveratrol and trans-mulberroside A (MUST bio-technology Co., Ltd.

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Chengdu, China) were determined using a Waters 2487 HPLC system (Waters,

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Milford, MA, USA) equipped with a reverse-phase 5 µm C18 column (4.6 × 250 mm)

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(InertSustain, Tokyo, Japan). The separation temperature was 30°C, and the detection

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occurred at 330 nm. The flow rate was 0.8 mL/min, and 10 µL samples were injected.

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The mobile phases were (A) acetonitrile and (B) 0.1% aqueous phosphoric acid. The

a

0.22

µm

filter.

Trans-resveratrol

(Sangon,

6


Shanghai,

China),

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solvent gradient elution program was as follows: 0–10 min, 11% A, 89% B; 10–15

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min, 11–24% A, 89–76% B; 15–55 min, 24% A, 76% B; and 55–60 min, 24–11% A,

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76–89% B. In addition, an ultra-performance liquid chromatography–mass

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spectrometer (UPLC–MS) was also used. A Waters ACQuity UPLC I class system

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was equipped with a BEH C18 1.7 µm column and TUV/QDa detector (Waters).

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Negative scanning mass spectra were acquired over the range from 100 to 600 m/z.

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Establishment of a co-expression system and fermentation tests

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We used the co-expression methods described previously, with a few

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modifications.17 Primers for vector construction are shown in Table S3. Ma4CL2

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carried a BamHI site at its 5'-end, and PstI and XhoI sites at its 3'-end. It was first

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cloned into the pET28a(+) (Novagen) at BamHI/XhoI sites, which resulted in the

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pET4CL2 cassette. The pET28a(+) fragment from AGATCTCGATCCCGCGAA to

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GGATCC (the BamHI site) was cloned and mutated from AGATCT to CTGCAG (a

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PstI site) and mutated from GGATCC (the BamHI site) to GGTACC (the KpnI site). It

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was then linked to the 5'-end of MaSTS3 by fusion PCR. The digested fusion fragment

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was inserted into the PstI/XhoI sites of pET4CL2, resulting in pET4CL-T-STS. Thus,

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MaSTS3 in pET4CL-T-STS could be replaced by other genes that carried KpnI/XhoI

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sites. Then, we introduced a gene of uncertain function (accession: ALS20361), which

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was identified as a MaSTS by bioinformatics methods, and we named it as MaSTS′ in

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this study. Thus, Ma4CL2 and MaSTS3/MaSTS′ were preceded by a T7 promoter/lac

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operator and a ribosome-binding site (Figure 2a, b).

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The co-expression plasmid was transformed into E. coli BL21(DE3) pLysS 7



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(Novagen). The cells were incubated in shake flasks on a rotary shaker at 37°C until

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the OD600 reached ~0.6, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside

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(IPTG), and cultivated at 25°C for 5 h to produce protein. Then, the cells were

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harvested at 5,000 ×g for 5 min and resuspended in M9 medium containing 1 mM

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p-coumaric acid, 0.1 mM malonyl-CoA lithium salt, 0.1 mM IPTG and 50 mg/L

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kanamycin (Kan). Fermentation was continued for 60 h at 25°C. The cells in the

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fermentation liquor were lysed by sonication and concentrated by a rotary evaporator.

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Subsequently, it was extracted three times with equal volumes of ethyl acetate and

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dried by a rotary evaporator. It was then dissolved in methanol and analyzed by HPLC

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and UPLC-MS.

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Construction and transformation of the recombinant plasmid for tobacco

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transformations

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Full-length MaSTS3 cDNA fragments were cloned into the BamHI and SpeI sites

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of pLGNL (conserved in our laboratory), which contained the cauliflower mosaic

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virus 35S promoter, to generate a pLGNL-MaSTS3 over-expression cassette (Figure

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2c). For tobacco transformations, Agrobacterium tumefaciens containing the plasmid

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was transformed into tobacco (Nicotiana tabacum L.) plants using the leaf disc

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method.18 Transgenic tobacco was selected on 1/2 Murashige and Skoog medium

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containing 50 mg/L Kan. The positive plants were confirmed by β-glucuronidase

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staining, and genomic PCR and qRT-PCR analyses.

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Physiological and abiotic stress tolerance analysis of transgenic tobaccos

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The T1 transgenic lines and wild type (WT) tobacco samples were cultivated in a 8


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climate chamber (27°C, 14 h day/10 h night, 8,000 Lx). After growing ~3 weeks, they

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were subjected to 40°C, salt (400 mM NaCl) and drought [20% polyethylene glycol

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(PEG) 6000] stresses, independently, for ~2 weeks. Leaves from the same positions

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were collected for free proline and malonaldehyde (MDA) content measurements. The

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proline content analysis was performed according to a previously published method19

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in which 0.5 g of fresh leaves were cut into small pieces, homogenized in 5 mL of 3%

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sulfosalicylic acid, boiled 10 min and centrifuged at 4,000 ×g for 10 min. A 2 mL

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extract was incubated with 2 mL of ninhydrin reagent, which contained 2.5% (w/v)

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ninhydrin, 40% 6 M phosphoric acid and 60% (v/v) glacial acetic acid, and 2 mL of

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glacial acetic acid, and then it was continuously boiled for 30 min. After the reaction

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was terminated in an ice bath, 4 mL of toluene was added and vortexed. The reaction

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mixture was centrifuged at 3,000 ×g for 5 min, and the absorbance of the supernatant

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at 520 nm was determined using a spectrometer (TECHCOMP, Shanghai, China).

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The MDA content was determined as described by previous research.20 Briefly,

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0.5 g of fresh tobacco leaves was homogenized in 5 mL 10% trichloroacetic acid and

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centrifuged at 12,000 ×g for 10 min. Then, 2 mL 0.6% thiobarbituric acid dissolved in

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10% trichloroacetic acid was added to 2 mL of the supernatant. The mixture was

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boiled for 20 min and then terminated in an ice bath. Then, it was centrifuged at

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12,000 ×g for 3 min, and the absorbance of the supernatant was determined at 532,

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450 and 600 nm. The MDA content was calculated as described previously.20

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To analyze the effects of MaSTS3 on other genes in transgenic tobacco (N.

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tabacum), the transcription of Nt4CL1 (U50845), Nt4CL2 (U50846) and NtCHS 9



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(XM_016638898 and XM_016634418) was determined by qRT-PCR (primers are

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listed in Table S4). In addition, to analyze the metabolites of transgenic tobacco,

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extracts were prepared from each line and extracted using the method described

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above.

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Statistical analyses

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All experiments were performed in triplicate, and the results were expressed as

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means ± standard deviation (SD). The statistical analysis was performed using SPSS

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Statistics 18.0 (SPSS Inc., Chicago, IL, USA) with Duncan's multiple range tests.

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Figures were drawn using OriginPro 7.5 (OriginLab, Northampton, MA, USA). Mean

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values that were significantly different within treatments were designated with an

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asterisk.

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Results

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Expression of MaSTS genes in different tissues and the contents of three stilbenes

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in different tissues

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Based on a multiple sequence alignment against the Morus genome and NCBI

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databases, four candidate STS genes were selected, and they were identified by

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cloning their corresponding cDNAs, and named as MaSTS1, MaSTS2, MaSTS3 and

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MaSTS4, respectively (File S1). To investigate the relationships between the

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expression levels of MaSTS genes and the accumulations of stilbenes, transcription

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levels of MaSTS genes were quantitatively measured by qRT-PCR, and the contents of

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the three stilbenes were determined by HPLC (Figure 3). An expression analysis

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revealed that MaSTS genes were expressed in various mulberry tissues, but exhibited 10


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significant differences in their magnitudes of expression (Figure 4, a1). MaSTS1 was

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mainly expressed in root bark, stem bark and old leaves, while MaSTS3 was highly

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expressed in root bark, stem bark and branch bark. MaSTS2 and MaSTS4 were highly

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expressed in root bark. Additionally, all of the MaSTS genes were expressed at

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relatively low levels in young leaves. Using the method above, the contents of the

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three stilbenes were determined. The mulberroside A content was greater than the

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oxyresveratrol and resveratrol contents in all of the selected tissues (Figure 4, a2–a4).

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The resveratrol content showed a similar pattern as mulberroside A, although

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resveratrol was not detected in young leaves (Figure 4, a2). Oxyresveratrol had a high

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content in root bark and branch bark (Figure 4, a3). Moreover, mulberroside A was the

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most abundant in root bark, reaching 9.09 mg/g of dry weight (mg/g DW), followed

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by stem bark and branch bark (Figure 4, a4).

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The dynamics of the three stilbene’s contents during mulberry fruit development

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from S1 to S7 (Figure 4, b1) were determined. As in other tissues, mulberroside A was

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the main stilbene form present. However, resveratrol existed throughout fruit

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development and reached its maximum of 0.06 (mg/g DW) at S7 (Figure 4, b2). The

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mulberroside A content’s trend was identical to that of oxyresveratrol, and they

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reached their maximum levels of 0.802 (mg/g DW) and 0.084 (mg/g DW),

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respectively, at S5 (Figure 4, b3 and b4). The four MaSTS showed expression

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differences during fruit development (Figure 4, c1–c4). MaSTS1 increased with the

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fruit developmental stage and reached its maximum at S7, although it was transiently

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down-regulated at S5 (Figure 4, c1). MaSTS2 and MaSTS3 were lowly expressed at 11



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the early stages, but their expression increased sharply and reached maximum levels

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at S6 and S4, respectively (Figure 4, c2 and c3). MaSTS4 performed irregularly and

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peaked at S3 (Figure 4, c4).

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Fermentation test

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The recombined plasmids of pET4CL2, pET4CL-T-STS3 and pET4CL-T-STS′

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were fermented in E. coli BL21 (Figure 5a). The color of the fermentation broth

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showed obvious differences after 60 h at 25°C. The broth of pET4CL2 was milky

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white, while pET4CL-T-STS3 was canary yellow and pET4CL-T-STS′ was yellow

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(Figure 5b), indicating that the function of MaSTS3 was different from that of MaSTS′.

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In addition, about 0.187 mg/L trans-resveratrol was produced in the pET4CL-T-STS3

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fermentation broth, and naringenin was detected in the pET4CL-T-STS′ ethyl acetate

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extract (Figure 5c).

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Dynamic expression of MaSTS genes under abiotic stresses

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MaSTS genes were sensitive to ABA treatments. MaSTS1 and MaSTS2 showed a

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“W-shaped” expression pattern, reached their minimum values at 1 h and 12 h,

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respectively, and peaked at 3 h and 24 h, respectively (Figure 6 a, b). MaSTS3 and

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MaSTS4 showed a completely different expression pattern. MaSTS3 decreased

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immediately and maintained a low expression level compared with the control (Figure

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6 c). However, MaSTS4 increased immediately after ABA treatment and maintained a

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high level (Figure 6 d).

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Under the SA treatment, MaSTS1, MaSTS2 and MaSTS3 showed similar

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expression patterns, peaking at 6 h and then being down-regulated (Figure 6 a–c). 12


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However, MaSTS4 showed a different pattern, reaching its maximum level at 3 h

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(Figure 6, d).

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For the NaCl treatment, the four MaSTS genes showed different reactions.

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MaSTS1 and MaSTS2 increased immediately and peaked at 3 h and 1 h, respectively

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(Figure 6 a, b). Then, MaSTS1 decreased gently, while MaSTS2 increased once again

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after 12 h and reached its maximum at 24 h. MaSTS3 showed a “U-shaped”

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expression pattern, which decreased sharply before 12 h, then increased continuously

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and finally reached a maximum at 24 h (Figure 6 c). However, MaSTS4 was

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expressed at a low level for 12 h but then greatly increased, reaching a maximum that

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was 14.03-fold greater than that of the control at 24 h (Figure 6 d).

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The MaSTS genes exhibited various responses to the wounding treatment.

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MaSTS1 and MaSTS2 showed similar expression patterns and peaked at 6 h. Their

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peak values were 52.93- and 50.91-fold greater, respectively, than that of the control

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(Figure 6 e). Interestingly, MaSTS3 and MaSTS4 also showed similar expression

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patterns but reached maximum levels at 12 h. However, MaSTS4’s level was more

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than 61.09-fold greater than that of the control, while MaSTS3’s level was only

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9.46-fold greater (Figure 6 e).

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Analysis of other genes regulated by MaSTS3 and the enhanced tolerance of

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transgenic tobacco to multiple abiotic stresses

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STS and CHS in plants used the same substrates (Figure 1); therefore, we

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selected upstream and downstream genes of CHS in N. tabacum to determine the

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genes that were regulated by MaSTS3. The expression level of MaSTS3 in six 13



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transgenic lines was quantified using qRT-PCR. Three differently expressing

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transgenic lines were selected for the next test. The qRT-PCR showed the expression

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levels of MaSTS3 were significantly higher than WT (Figure 7, a1). Nt4CL1 in

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over-expression line 4 (OE4) and OE5 was up-regulated, but there was no obvious

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change in OE6 (Figure 7, a2). Interestingly, Nt4CL2 in WT was significantly

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up-regulated, and its expression trend was similar to that of MaSTS3 (Figure 7, a3).

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However, the expression levels of NtCHS in OE4 and OE6 were not different from

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that of WT, while the expression of NtCHS in OE5 increased (Figure 7, a4).

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Additionally, the total flavonoid contents in transgenic tobacco lines were also

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determined. The flower color was not significantly different between the transgenic

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tobacco and the WT (Figure 7, b1). The total flavonoid contents in transgenic tobacco

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lines showed less obvious reductions compared with the control group (Figure 7, b2).

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Additionally, HPLC chromatograms revealed trans-resveratrol was accumulated, with

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45.167 (µg/g FW), which was not presented in the WT (Figure 7, b3).

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Three transgenic tobacco lines and WT seedlings were subjected to various

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abiotic stresses to characterize the functions of MaSTS3 under NaCl, heat and PEG

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stress. After ~2-week treatments, transgenic lines and WT showed some differences

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compared with the control (Figure 7, b4 and c1). WT showed more sensitivity than

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transgenic lines to salt and heat treatments, and the leaves started to turn yellow.

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However, there were no evident morphological differences between the transgenic

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line and the WT seedlings under drought stress. The proline contents of OE4 and OE5

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were higher than that of WT under the PEG treatment, although OE6 showed no 14


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significant change compared with WT (Figure 7, c2). Additionally, the proline

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contents of all of the transgenic lines were higher than that of WT under the NaCl and

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heat treatments (Figure 7, c3 and c4). The MDA content was also determined. All of

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the transgenic lines had lower MDA contents than the WT under PEG and NaCl

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treatments (Figure 7, d1–d3). However, the MDA contents of OE5 and OE6 were

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higher than WT under the heat treatment (Figure 7, d4).

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Discussion

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Mulberry is widely distributed in China and contains more phenolic and flavonoid

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compounds than some other fruits and vegetables.21 The root, branch bark and leaves

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of mulberry are used as traditional medicinal materials, and the fruit is used as

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nutritional foodstuff.22 In addition, mulberry is well known for its capacity to resist

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harsh environments. It can grow in areas severely affected by desertification,

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including sand damage, drought and saline stress.

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Although all of the MaSTS genes were detected in selected tissues, they showed

331

obvious differences. MaSTS3 was highly expressed in root bark, stem bark and branch

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bark. In addition, the expression levels of MaSTS3 were in accordance with the

333

stilbene contents of select tissues. The expression trend of MaSTS3 was also similar to

334

the trend of the stilbene contents during fruit developmental stages. Thus, MaSTS3

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was the major STS gene in mulberry.

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Mulberroside A was the major stilbene in mulberry, followed by oxyresveratrol.

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Nevertheless, the parent nucleus had the lowest level of trihydroxystilbene resveratrol

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in the different tissues. This was similar to the results of other studies.23-25 The major 15



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stilbene, 2,3,5,4'-tetra-hydroxy-stilbene-2-O-β-D-glucoside, was abundant in the

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rhizomes and old stems of F. multiflora.24 A similar situation occurred in the genus

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Picea, in which the stilbene glucosides astringin and isorhapontin were at high

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concentrations in the roots and bark.23,25 In vitro and in vivo assays revealed that STS

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catalyzed the condensation of one 4-coumaroyl-CoA and three molecules of

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malonyl-CoA to form the trihydroxystilbene resveratrol.11,23 However, over-expressed

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PaSTS1 in transgenic Norway spruce showed that significantly higher amounts of the

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tetrahydroxystilbene glycosides isorhapontin and astringin were produced.23 Similarly,

347

over-expressed SbSTS in Arabidopsis tt4 mutants could lead to the accumulation of

348

cis-resveratrol glucoside (piceid), which is the major stilbene in the transgenic lines.26

349

Those results suggested that the first step in the biosynthesis of stilbenes in plants was

350

the formation of resveratrol, which was then further modified by hydroxylation,

351

O-methylation and O-glucosylation.23,25,26 Additionally, previous studies showed that

352

trans-resveratrol accumulates in the early stages of seedling roots. Additionally, the

353

contents of trans-oxyresveratrol and trans-mulberroside A increased while resveratrol

354

decreased during mulberry early development.27 Therefore, we speculate that

355

oxyresveratrol and mulberroside A are probably transformed from resveratrol through

356

oxidation, O-methylation or other derivatizations in mulberry.

357

The resveratrol STS genes were originally described in peanuts and grapes,11,28

358

which accumulate elevated levels of the stilbene following biotic and abiotic stresses,

359

including protecting plants against fungal invasions.23,29 SA is an important signal

360

molecule in the plant host’s defense response process, and the endogenous SA levels 16


Page 16 of 36

Page 17 of 36


361

were involved in the activation of pathogenesis-related gene expression.30 However,

362

all of the MaSTS genes participated in the up/down-regulatory processes, indicating

363

that MaSTS genes are involved in feedback regulation when stimulated by SA. This is

364

similar to previous research on the expression pattern of Vitis’ STS under powdery

365

mildew infection.28,29 SA signaling in plant defense is part of a complex network,

366

including feedback loops.31-33 Therefore, we speculated that exogenous SA influences

367

the endogenous SA levels at the first step, then triggers MaSTS feedback loops in the

368

next process.

369

As another important signaling molecule involved in the plant immune response,

370

ABA improves plant tolerances to salt and dehydration.34-36 In plant cells, salt and

371

drought lead to increased reactive oxygen species levels, which then stimulate the

372

synthesis and accumulation of ABA in roots.37,38 Here, MaSTS genes were sensitive to

373

ABA stress. In particular, MaSTS3 showed a complicated expression level that was

374

always down-regulated while MaSTS4 was always up-regulated. The recent

375

immunolocalization of STS revealed that stilbene biosynthesis takes place within the

376

cell wall.39,40 Interestingly, the upstream Ma4CLs were up-regulated significantly to

377

synthesize lignin when mulberry were stimulated by ABA, even though Ma4CL was

378

related to flavonoid synthesis in normal times.17 The process reduced the substrate

379

level of MaSTS, which may explain the genes’ down-regulation. In addition, the

380

expression of MaSTS genes under NaCl stress differed from that under ABA stress.

381

This implies that MaSTS genes respond to salt stress through an ABA-independent

382

signal transduction pathway. 17



383

When plants suffer from mechanical damage, the cell walls of the damaged sites

384

are first strengthened by crosslinking proteins to prevent dehydration and possible

385

pathogen infections, and then phenylpropanoid derivatives are required to be

386

synthesized in the subsequent step.17,41 As an outstanding phytoalexin, stilbene is a

387

potential fungicidal agent.4,25 This may explain why the wounding treatment led to a

388

high induction of MaSTS genes.

389

STS was frequently used to modify plant secondary metabolism to elevate the

390

self-defense capacity or the nutritional quality of crops.6,42,43 However, most reports

391

focused on increased tolerance against microbial pathogens.43,44 Transgenic tobacco

392

improved the tolerance capability, at different magnitudes, to PEG and NaCl

393

treatments, as evidenced by the higher proline and the lower MDA contents compared

394

with in WT. Additionally, over-expressed MaSTS3 genes modified the transcription of

395

endogenous genes in transgenic tobacco. Interestingly, the expression pattern of

396

Nt4CL2 was in accordance with that of MaSTS3. Thus, Nt4CL2 was associated with

397

stilbene synthesis, and the result was consistent with previous research showing that

398

Nt4CL2 was more likely to biosynthesize non-lignins.45 Additionally, NtCHS

399

expression was distinctly different among the transgenic lines, with only OE5 being

400

up-regulated, while OE4 and OE6 remained unchanged, compared with WT. As

401

described previously, CHS utilizes the same substrates as STS;11 therefore, the

402

over-expression of MaSTS3 may result in foreign MaSTS3 competing with

403

endogenous NtCHS for a limited substrate. However, the expression levels of

404

MaSTS3 in OE4 and OE6 were higher than in WT but lower than in OE5, which 18


Page 18 of 36

Page 19 of 36


405

meant that the substrate content was enough for both MaSTS3 and NtCHS. This

406

would eliminate the competition between MaSTS3 and NtCHS. Previously, plants

407

were transformed with STS genes from different plants, leading to an accumulation of

408

resveratrol or its derivatives. For instance, transformations of Arachis hypogea44 and

409

grapevine46 STS genes led to the accumulation of resveratrol in tobacco. Other

410

STS-coding genes have also been transformed to Arabidopsis thaliana, such as

411

SbSTS1 (Sorghum bicolor)11 and PcRS (Polygonum cuspidatum),43 which resulted in

412

piceid accumulation. As expected, HPLC chromatograms of the extracts from

413

transgenic tobaccos had obvious peaks that were not detected in the extracts of the

414

WT plants, and UPLC–MS detected a resveratrol (227 m/z) signal. This indicated that

415

trans-resveratrol was accumulated after MaSTS3 over-expression.

416

Resveratrol has been recognized for its benefits to human health. Currently, an

417

increasing demand for resveratrol for cosmetic, nutraceutical, and putative

418

pharmaceutical uses makes its production from a sustainable source a necessity.47

419

However, because of the low content in natural products, the environmental pollution

420

and disruption caused by the excessive use of raw materials has become increasingly

421

serious.17,48 It is necessary to establish a reliable alternative method of resveratrol

422

production under controlled conditions.47 As indicated by the stilbene contents

423

determined above, there is a highly efficient stilbene production system in mulberry,

424

which can be simulated in vitro to produce resveratrol. We built a recombinant

425

plasmid harboring two promoter/lac operators to independently regulate Ma4CL2 and

426

MaSTS3. Although the use of biotechnology through recombinant bacteria has been 19



427

reported,49,50 the methods did not suit this study because a soluble Ma4CL protein

428

could not be obtained.17 Additionally, CHS is highly similar to STS, leading to

429

numerous plant STS being annotated as CHS genes in different public databases.5 The

430

most used method for identifying CHS/STS is the determination of the product by

431

expressing it in transgenic plants or by enzymatic reactions in vitro.11,26 However, the

432

substrates are not easy to produce. Thus, we established this system to conveniently

433

distinguish CHS/STS. We improved the recombinant plasmid, adding a KpnI cloning

434

site at the 5′ of MaSTS3 that easily allows the insertion of other STS/CHSs. In our

435

previous study, we identified MaSTS′ genes by bioinformatics method. However, our

436

data demonstrate that ALS20361, in fact, encodes a CHS enzyme.

437

‘Jialing No. 40’, which is a new polyploidy variety of fruit-producing mulberry,

438

was bred in our laboratory in recent years,22 and it possesses a high product yield of

439

good quality fruit and leaves. In conclusion, the fruit of ‘Jialing No. 40’ not only have

440

a high yield but are also rich in stilbenes. Four MaSTS genes were identified in this

441

study, and the stress tests suggested that MaSTS3 genes participate in a series of

442

abiotic and biotic stresses. Genetic transformation tests indicated that MaSTS3 could

443

accumulate trans-resveratrol and improve tobacco tolerance to abiotic stresses.

444

Furthermore, the novel co-expression system of Ma4CL2 and MaSTS3 was

445

established, and trans-resveratrol was successful produced by fermentation. In

446

particular, the co-expression system could be used to identify the functions of

447

CHS/STS. This study provides the basis for future research on the stilbene metabolic

448

pathway in mulberry. 20


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449

Abbreviations Used Ma Nt STS CHS CHI 4CL RT-PCR qRT-PCR ABA SA WT OE DW FW IPTG Kan

Morus atropurpurea Nicotiana tabacum stilbene synthase chalcone synthase chalcone isomerase 4-coumarate:CoA ligase reverse transcription polymerase chain reaction real-time quantitative polymerase chain reaction abscisic acid salicylic acid wild type over-expression dry weight fresh weight isopropyl-β-D-thiogalactopyranoside kanamycin

450

451 452

Acknowledgements We thank associate professor Li Xu and Haipeng Lu for providing HPLC

453

detection, and Hu Chen for providing UPLC–MS detection.

454

Funding

455

The work was funded by the China Special Fund for Agro-scientific Research in

456

the Public Interest (grant No. 201403064), Fundamental Research Funds for the

457

Central Universities (grant No. XDJK2016D024), the China Agriculture Research

458

System (grant No.CARS-22), and the National Natural Science Foundation of China

459

(grant No. 31360190)

460

Notes 21



461

462 463 464

The authors declare no competing financial interest.

Supporting Information Table S1. The primers used to isolate the MaSTS genes in Jialing No.40/ Guiyou62.

465

Table S2. qRT-PCR Primers for MaSTS genes.

466

Table S3. Oligonucleotides for vector construction in this study.

467

Table S4. qRT-PCR Primers for transgenic tobaccos.

468

File S1. The cDNA sequences of MaSTS genes isolated from Jialing No.40/

469

Guiyou No. 62.

470 471 472 473 474 475 476 477 478 479 480 481 482 22


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483

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614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656

14, 613-21.

26


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657

Figure captions

658

Fig. 1. Biosynthetic pathways of resveratrol and chalcone. The enzymes of general

659

phenylpropanoid metabolism, which are connected by black arrows, consist of

660

phenylalanine ammonia-lyase (PAL), cinnamic 4-hydroxylase (C4H), and 4CL.

661

STS and CHS used the same substrates, and naringenin chalcone is usually

662

spontaneously converted to naringenin in vitro.

663

Fig. 2. Schematic representation of the strategy used for constructing the

664

co-expression vector pET4CL-T-STS (a and b), 28 indicates the fragment cloned

665

from pET28a(+); Construction the vector for tobacco transformations (c).

666

Fig. 3. Chromatograms of three standard stilbenes and mulberry extractions. (a)

667

standard chromatogram of the three stilbenes. (b) and (c) chromatograms of root

668

bark and fruit extractions, respectively.

669

Fig. 4. Expression of MaSTS genes (a1) and the content of stilbenes in root bark (RB),

670

stem bark (SB), branch bark (BB), old leaf (OL) and young leaf (YL) (a2-a4);

671

b1*, images [previously published (17)] indicate fruit development from S1 to

672

S7. In addition, the contents of stilbenes (b2–b4) and the expression levels of

673

MaSTS genes (c1–c4) in fruits are shown, ND means that no resveratrol was

674

detected.

675

Fig. 5. Biosynthesis routes of resveratrol constructed in recombinant E. coli (a) and

676

fermentation broth (b); (c1–c3) chromatograms of pET4CL, pET4CL-T-STS3 and

677

pET4CL-T-STS′, respectively.

678

Fig. 6. The relative expression levels of MaSTS genes under a series of treatments. a, 27



679

b, c and d indicate the expression levels of four MaSTS genes under ABA, SA,

680

NaCl and wounding treatments, respectively. “*” indicates p < 0.05, “**”

681

indicates p < 0.01.

682

Fig. 7. Analysis of the expression levels of MaSTS3, Nt4CL1, Nt4CL2 and NtCHS in

683

transgenic tobaccos (a1–a4); The total flavonoid contents of transgenic tobacco

684

flowers (b1 and b2), chromatograms of tobacco extracts (b3) and phenotypes of

685

WT and transgenic lines after two week treatments (b4). Proline (c1–c4) and

686

MDA contents (d1–d4) for control, PEG, NaCl and heat treatments, respectively.

687

“*” indicates p < 0.05, “**” indicates p < 0.01.

28


Page 28 of 36

Page 29 of 36


Figure 1. Biosynthetic pathways of resveratrol and chalcone. The enzymes of general phenylpropanoid metabolism, which are connected by black arrows, consist of phenylalanine ammonia-lyase (PAL), cinnamic 4-hydroxylase (C4H), and 4CL. STS and CHS used the same substrates, and naringenin chalcone is usually spontaneously converted to naringenin in vitro. 190x155mm (300 x 300 DPI)



Figure 2. Schematic representation of the strategy used for constructing the co-expression vector pET4CL-TSTS (a and b), 28 indicates the fragment cloned from pET28a(+); Construction the vector for tobacco transformations (c). 156x93mm (300 x 300 DPI)


Page 30 of 36

Page 31 of 36


Figure 3. Chromatograms of three standard stilbenes and mulberry extractions. (a) standard chromatogram of the three stilbenes. (b) and (c) chromatograms of root bark and fruit extractions, respectively. 142x155mm (300 x 300 DPI)



Figure 4. Expression of MaSTS genes (a1) and the content of stilbenes in root bark (RB), stem bark (SB), branch bark (BB), old leaf (OL) and young leaf (YL) (a2-a4); b1*, images [previously published (17)] indicate fruit development from S1 to S7. In addition, the contents of stilbenes (b2–b4) and the expression levels of MaSTS genes (c1–c4) in fruits are shown, ND means that no resveratrol was detected. 167x93mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36


Figure 5. Biosynthesis routes of resveratrol constructed in recombinant E. coli (a) and fermentation broth (b); (c1–c3) chromatograms of pET4CL, pET4CL-T-STS3 and pET4CL-T-STS′, respectively. 179x87mm (300 x 300 DPI)



Figure 6. The relative expression levels of MaSTS genes under a series of treatments. a, b, c and d indicate the expression levels of four MaSTS genes under ABA, SA, NaCl and wounding treatments, respectively. “*” indicates p < 0.05, “**” indicates p < 0.01. 141x155mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36


Figure 7. Analysis of the expression levels of MaSTS3, Nt4CL1, Nt4CL2 and NtCHS in transgenic tobaccos (a1–a4); The total flavonoid contents of transgenic tobacco flowers (b1 and b2), chromatograms of tobacco extracts (b3) and phenotypes of WT and transgenic lines after two week treatments (b4). Proline (c1–c4) and MDA contents (d1–d4) for control, PEG, NaCl and heat treatments, respectively. “*” indicates p < 0.05, “**” indicates p < 0.01. 196x170mm (300 x 300 DPI)



table of contents (TOC) 56x47mm (300 x 300 DPI)


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Characterization of Stilbene Synthase Genes in mulberry (Morus

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