Development of Marker-Free Transgenic Potato Tubers Enriched in

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Development of marker-free transgenic potato tubers enriched in caffeoyl quinic acids and flavonols YANG LI, Wenzhao Tang, Jing Chen, Ru Jia, Lianjie Ma, Shaoli Wang, Jiao Wang, Xiangling Shen, Zhaohui Chu, Changxiang Zhu, and Xinhua Ding J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00270 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on April 2, 2016

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Journal of Agricultural and Food Chemistry

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Development of marker-free transgenic potato

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tubers enriched in caffeoyl quinic acids and

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flavonols

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YANG LI†, WENZHAO TANG‡, JING CHEN†, RU JIA†, LIANJIE MA†, SHAOLI WANG†, JIAO WANG†, XIANGLING SHEN#, ZHAOHUI CHU†, CHANGXIANG

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ZHU*†Φ, XINHUA DING*†

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7 8 †

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State Key Laboratory of Crop Biology, Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, Shandong Agricultural University, Taian 271018, Shandong, PR China

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‡

9 10

13 14 15 16

Key Laboratory for Rare Disease of Shandong Province, Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062, Shandong, PR China Φ

Shandong YuTai Biotechnology Company, Taian 271018, Shandong, PR China

#

Biotechnology Research Center, China Three Gorges University, Yichang City 443002, Hubei, PR China

17 18

AUTHOR INFORMATION

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Corresponding Authors

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*Phone: 86-538-8245569. Fax: 86-538-8249913. E-mail: [email protected]

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*Phone:86-538-8241245. Fax: 86-538-8249913. E-mail: [email protected]

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Notes

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The authors declare no competing financial interest.

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ACS Paragon Plus Environment


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ABSTRACT

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Potato (Solanum tuberosum L.) is a major crop worldwide that meets human

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economic and nutritional requirements. Potato has several advantages over other

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crops: easy to cultivate and store, cheap to consume and rich in a variety of secondary

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metabolites. In this study, we generated three marker-free transgenic potato lines that

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expressed the Arabidopsis thaliana flavonol-specific transcriptional activator

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AtMYB12 driven by the tuber-specific promoter Patatin. Marker-free potato tubers

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displayed increased amounts of caffeoyl quinic acids (CQAs) (3.35-fold increases on

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average) and flavonols (4.50-fold increase on average). Concentrations of these

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metabolites were associated with the enhanced expression of genes in the CQA and

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flavonol biosynthesis pathways. Accumulation of CQAs and flavonols resulted in

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two-fold higher antioxidant capacity compared to wild-type potatoes. Tubers from

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these marker-free transgenic potatoes have therefore improved antioxidant properties.

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KEYWORDS: marker-free transgenic potato, phenylpropanoid improved, antioxidant capacity,

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MYB transcription factors


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INTRODUCTION

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Among the six major world crops (maize, rice, wheat, potato, cassava and soybean),

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potato (Solanum tuberosum L.) is the fourth most cultivated.1 In Asia, a majority of

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the potato harvest is used to make bread, buns, noodles and flour. In Europe, over

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50% is processed into chips, fried foods and other similar products.2 Food shortages

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remain a global problem due to limited cultivatable land and water, the influence of

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climate change, and the difficulty of improving the productivities of crops.

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Fortunately, the space for potential improvements in potato productivity remains large.

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As a result, potatoes are purported to be a promising solution to future food crises.

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The consumption of fruits and vegetables reduces the risk of certain diseases and

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improves human health,3 and potato is a critical vegetable that is highly consumed in

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many countries. In an attempt to ensure the security of China’s food supply and to

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meet the nutritional and health demands of the country, the Staple Crop of Potato

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Development Strategy Symposium officially declared potato as a staple crop in 2015.

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Potato is a rich source of vitamins C, B1, B3 and B6 as well as phenolics and

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flavonoids.4 Of these chemicals, phenolics and flavonoids are considered as the major

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antioxidant compounds in potatoes and other vegetables.

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Caffeoyl quinic acids (CQAs) are the most abundant phenolic acids in

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potatoes,4 making up approximately 80 % of the total phenolic content. CQAs are

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important nutritional compounds due to their high bioavailability: CQAs can be

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absorbed by the intestines and exert their antioxidant potential following

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absorption.5-7 Four CQA isomers exist, each with different caffeoyl acid/quinic acid

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ester bond locations: 1-CQA, 3-CQA, 5-CQA (chlorogenic acid, CGA) and 4-CQA.

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However, only three types of CQAs have been observed (3-CQA, 4-CQA and 5-CQA)

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in potato tubers. The concentrations of the 3-CQA and 4-CQA isomers are relatively



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low in potatoes.1,8 The 5-CQA (CGA) is the major antioxidant in potatoes, and this

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compound has been shown to be beneficial to human health. CGA has the ability to

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inhibit carcinogenesis in the colon and liver, and protect cells against oxidative stress

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as well. In addition, CGA has been shown to reduce the risk of cardiovascular

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disease.9,10 It has also been suggested that CGA can cause weight loss via inhibition of

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preadipocyte proliferation.11,12 The 1-CQA has been shown to be a potential inhibitor

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of the NF-ĸB pathway, demonstrating the anticarcinogenic properties and dietary

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phytochemical potential of 1-CQA. This CQA influences not only the early stages of

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the immune response but also each stage of the inflammatory process.13,14 The 3-CQA

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is considered one of the richest sources of polyphenols in the human diet and has the

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capacity to prevent liver diseases, hyperlipidemic diseases and obesity.15,16 The

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4-CQA plays a role in the anti-inflammatory activity of Hibiscus sabdariffa leaves.17

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CQAs are synthesized via the phenylpropanoid pathway,4 and many intermediates in

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the pathway are used as precursors in the flavonoid pathway.18 Three CQA

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biosynthetic pathways have been identified: (1) CQAs can be synthesized from

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caffeoyl-CoA and quinic acid by hydroxycinnamoyl-CoA quinate hydroxycinnamoyl

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transferase (HQT) in potatoes, tomatoes and tobacco (route 1 in Figure 1);19,20 (2) in

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the species without HQT, CQA can be synthesized from quinic acid and caffeoyl

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glucoside via HCGQT (hydroxycinnamoyl ᴅ-glucose: quinate hydroxycinnamoyl

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transferase) (route 2 in Figure 1);21 (3) p-coumaroyl-CoA and quinic acid can by

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combined by hydroxycinnamoyl-CoA transferase (HCT) to form p-coumaroyl quinic

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acid, then acted by p-coumarate 3’-hydroxylase (C3’H) to form CQA.22,23

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Flavonoids, a major class of secondary metabolites, function as antioxidants

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with pharmacological properties: flavonoids have been shown to be anticarcinogenic

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and anti-inflammatory as well as inhibitors of platelet aggregation.24 Quercetin


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rutinoside, a flavonoid, scavenges radicals and inhibits acetylcholinesterase and

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butyrylcholinesterase to induce lipid peroxidation in brain homogenates.25

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Kaempferol rutinoside extract isolated from Angelica shikokiana significantly

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decreased amyloid β peptide-induced neurotoxicity and ThT fluorescence.26 Potato

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stem tubers contain nearly 10 types of flavonoids, primarily consisting of quercetin

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rutinoside, kaempferol rutinoside, quercetin glucosyl-glucoside rhamnoside and

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kaempferol glucosyl-glucoside rhamnoside (Figure 2). However, only small amounts

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of flavonols have been observed in potato tubers.1,27

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The MYB transcription factor family is ubiquitous in eukaryotes and widely

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distributed in plants. In recent years, several MYB transcription factors have been

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characterized and classified into groups based on the number of MYB DNA binding

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domains in their sequences. The R2R3-MYB family, which contains two repeats DNA

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binding domains, is the MYB family primarily responsible for regulating flavonol and

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CQA biosynthesis.28,29 AtMYB12, a R2R3-MYB factor in Arabidopsis thaliana,

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activates flavonol biosynthesis by upregulating the phenylpropanoid pathway (Figure

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2).30,31 The expression of AtMYB12 in tobacco, buckwheat and tomatoes resulted in

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increased flavonol production.32-34 CQA production was also increased significantly in

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tomatoes; however, CQAs cannot be synthesized in Arabidopsis thaliana.32-37 In

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general, the expression level of AtMYB12 is positively correlated with flavonol

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content.28, 31

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Currently, extensive administrative regulations from government departments;

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pressure of public opinion from media and the limited understanding from people,

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lead a query on controversy and potential risks of genetic food, though some genetic

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foods are full of antioxidants to improve human diets without fundamental changes.

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The selectable markers used during transformation processes are the subject of many



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of the public concerns regarding transgenic plants.38 Eliminating selectable markers

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could therefore increase the acceptability of transgenic techniques. Recently, several

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systems have been developed to generate marker-free transgenic plants, including

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site-specific recombination and transposition. However, these two approaches are

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all-consuming.39,40 A chemically induced system for generating marker-free transgenic

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plants was recently developed that allows markers to be removed via treatment with

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chemical inducers.41-43

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In this study, we isolated the flavonol-specific transcriptional activator

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AtMYB12 (cDNA) from Arabidopsis thaliana and transformed it into potato plants.

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Here, AtMYB12 is driven by Patatin, a potato tuber-specific expression promoter.

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After we obtained transgenic plants, the chemical inducer β-estradiol was used to

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generate marker-free transgenic lines. The major secondary metabolites produced in

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AtMYB12 transgenic potatoes were CQAs and flavonols. CQA contents in AtMYB12

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transgenic lines were significantly increased compared to wild-type controls: the

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concentrations of the four CQA isomers (1-CQA, 3-CQA, 4-CQA, 5-CQA) were

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3.35-fold higher on average. In addition, 1-CQA was observed in potato tubers for the

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first time. The contents of four major flavonols were increased to different levels

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(from 1.34- to 12.70-fold) separately.

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MATERIALS AND METHODS

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Constructs for transformation. The full-length cDNA of AtMYB12

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(AAC83586) was amplified from Arabidopsis thaliana Col-0 with AtMYB12 F1

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(5’-ACTTTTGTGGTCAGTGGAATA-3’) and R1 (5’-AACGGATCAATCAATATCA

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T-3’) using RT-PCR. This cDNA served as the template for further amplification with

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AtMYB12 F2 (5’-ATGGAATTCACTTTTGTGGTCAGTGGAATA-3’) and R2 (5’-AT


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GCTCGAGAACGGATCAATCAATATCAT-3’) with nested PCR. The PCR product

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was digested with XhoI and EcoRI, then ligated with similarly digested pBlueScript,

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generating the transitional vector pBS-AtMYB12. The Patatin promoter was amplified

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from Zhongshu NO.4 using the primer pair Patatin PROF1 (5’-ATGACTAGTGTTCA

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GTAATTGACCGGAGAC-3’; the SpeI site is underlined) and Patatin PROR1

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(5’-ATGGAATTCAATTTTGTTGGTGCTTTGAG-3’; the EcoRI site is underlined)

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according to the sequence deposited in GenBank (X03956). The amplified Patatin

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sequence was digested with SpeI and EcoRI, then ligated into SpeI/EcoRI digested

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pBS-AtMYB12 to produce the transitional vector pBS-Patatin::AtMYB12. The potato

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transformation vector pX6-Patatin::AtMYB12 was constructed by replacing GFP

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(double digested with XhoI and SpeI) in pX6 with Patatin::AtMYB12. This construct

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was

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Agrobacterium-mediated transformation into the internodal sections of potatoes (S.

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tuberosum cv. Desiree) was performed using a previously published method.44

transformed

into

A.

tumefaciens

strain

AGL1

by

electroporation.

115

Potato transformation media. M0: 1×MS, 0.2 mg/L NAA (1-naphthalenea-

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cetic acid), 0.02 mg/L GA3 (Gibberellin A3), 2.5 mg/L zeatin riboside, 20 g/L sucrose,

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8 g/L agar powder (pH: 5.7-5.8). M1: M0+500 mg/L cefotaxime (pH: 5.7-5.8). M2:

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1×MS, 0.02 mg/L NAA (1-naphthaleneacetic acid), 0.02 mg/L GA3 (Gibberellin A3),

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2 mg/L zeatin riboside, 500 mg/L cefotaxime, 50 mg/L kanamycin, 20 g/L sucrose, 8

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g/L agar powder (pH: 5.7-5.8). MR: 1×MS, 500 mg/L cefotaxime, 50 mg/L

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kanamycin, 20 g/L sucrose, 8 g/L agar powder (pH: 5.7-5.8).45

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Potato transformation and marker removal from AtMYB12-expressing Internode segments from Desiree potato cultivars grown 21-28 d in vitro

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potatoes.

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were cut into 0.5 cm explants. The explants were incubated in an Agrobacterium

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culture (OD=0.8) for 20 min with occasional agitation. The explants were then



126

removed and blotted on sterile filter paper. Then, the explants were plated on M0

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medium and incubated for 3 d in the dark at 18°C. Explants were then transferred to

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M1 medium and incubated for 12 d using man-made climate equipment with a 16 h

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light/8 h dark cycle at 21°C ± 1°C with a light intensity of 2000 lux.44 Following

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incubation, well-developed callus internode segments were transferred to M2 medium.

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Segments were regularly transferred to fresh M2 medium for 10-14 days. After

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approximately three rounds of selection, regeneration buds (0.2 cm) were excised and

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transferred to MR medium containing cefotaxime and kanamycin. After the weekly

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regenerated roots became strong, adventitious shoots (1 cm) from the transgenic lines

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were excised and transferred to M1 medium containing 3 µM β-estradiol and

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cefotaxime.45 Shoots that rooted well were transplanted to sterilized soil. The presence

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of full-length AtMYB12 DNA and NPT II DNA in AtMYB12 transgenic potato tubers

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was confirmed by PCR with the Patatin PROF1 (5’-ATGACTAGTGTTCAGTAATT

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GACCGGAGAC-3)/AtMYB12 R2 (5’-ATGCTCGAGAACGGATCAATCAATATCA

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T-3’) primer pair and the NPTII F1 (5’-AGCCAACAACGGTATGTCCTGAT-3’)/

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NPTII R1 (3’-TGAATGAACTGCAGGACGAG-3’) primer pair, respectively. Nine

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positive independent plants from three transgenic lines (1-1, 1-2, 1-3; 2-1, 2-2, 2-3;

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3-1, 3-2, 3-3) were obtained. Positive marker-free transgenic potato tubers were

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planted in a greenhouse set to 21°C ± 1°C.

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Semi-quantitative RT-PCR. Total RNA was isolated from 100 mg of plant

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tubers using TRIzol reagent according to the manufacturer’s instructions (Invitrogen,

147

Carlsbad, Calif., USA). The primers A12 F (5’-ACTTTTGTGGTCAGTGGAATA-3’)

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and A12 R (5’-GAGAACGGATCAATCAATATCAT-3’ ) were designed to detect. The

149

potato EF1α gene was used as a control and was amplified using the primers EF1α F

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(5’-ATTGGAAACGGATATGCTCCA-3’) and EF1α R (5’-TCCTTACCTGAACGCC


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TGTCA-3’). PCR thermal cycling conditions were as follows: 94°C for 3 min,

152

followed by 30 cycles of 94°C for 15 s, 58°C for 15 s, 72°C for 30 s and a final

153

extension at 72°C for 10 min. PCR products were confirmed using 1 % TAE-agarose

154

gel electrophoresis. All experiments were carried out with three biological repeats. Identification and quantification of phenylpropanoids.

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Phenylpropanoids

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in potato tubers were extracted from freeze-dried samples using 70 % methanol from

157

Sigma

158

LC-MS/MS and identificated by comparing the area of each individual peak with

159

calibration curves obtained from the pure compounds as Luo et al., 2008.32 CGA and

160

quercetin

161

(http://www.sigmaaldrich.com/).

162

Estrasynthese (http://www.extrasynthese.com).

(http://www.sigmaaldrich.com/).

rutinoside

(rutin)

Phenylpropanoids

standards Kaempferol

were

were

purchased

rutinoside

was

detected

by

from

Sigma

purchased

from

163

Samples were analyzed using an Agilent Technologies 1200 series HPLC

164

equipped with a diode array detector, a BDS HYPERSIL C18 column (Agilent

165

Technologies ZORBAX SB-C18 4.6*250 mm) and a mass detector (Agilent

166

Technologies, Waldbronn, Germany) for identification. The HPLC mobile phase was

167

a mix of water (containing 3 % acetonitrile and 10 % formic acid, solvent A) and

168

HPLC solvent B (40:50:10 water/acetonitrile/formic acid) at a flow rate of 1 mL min-1.

169

A gradient of increasing solvent B was used: beginning with 4% B, the solvent was

170

increased to 20 % B at 20 min, 40% B at 35 min, 60% B at 40 min, and 90% B at 45

171

min. At 55 min, the solvent was held isocratic at 4% B.46 UV chromatograms were

172

recorded at 325 nm. The mass spectrometer nebulizer gas was nitrogen. The pressure

173

and flow rate of the drying gas were set at 65 psi and 11 L min-1, respectively.1,32 The

174

heated capillary was maintained at 350 °C, and the capillary voltage was set at 4 kV.

175

Mass scan (MS) and daughter ion (MS/MS) spectra were acquired from m/z 100 up to



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176

m/z 1000. Collision-induced fragmentation experiments were performed in the ion

177

trap using helium as the collision gas and voltage ramping cycles from 0.3 to 2 V.

178

Mass spectrometry data were acquired in positive mode.

179

Quantitative real-time PCR (qRT-PCR). The concentrations and purities of

180

RNA samples were determined by UV absorbance spectrophotometry. First-strand

181

cDNA was synthesized using SuperQuick RT MasterMix (CWBio, Jiangsu, China)

182

following the manufacturer’s instructions. Transcription of phenylpropanoid

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biosynthetic genes was analyzed by quantitative PCR using gene-specific primers

184

(Supplementary Table 1).27 All amplifications of target genes were performed using

185

SYBR Premix Ex Taq (TaKaRa, Dalian, China). The EF1α gene was used as an

186

internal control and it was amplified under the same conditions using the specific

187

primers shown in Supplementary Table 1. Quantitative PCR was conducted on a

188

Bio-Rad iQTM5 Light Cycler as previously reported.46 Relative quantification

189

analysis was performed using a relative standard curve according to calculated

190

threshold values (Ct). We mixed plant tubers from all three marker-free lines for

191

detection of phenylpropanoid biosynthetic gene expression. All experiments were

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carried out with two biological and four technical replicates.

193

Total antioxidant activity. Freeze-dried potato tuber samples (50 mg) were

194

extracted with 70 % ethanol, and the antioxidant capacities of the extracts were

195

analyzed.

196

(3-ethylbenz-thiazoline-6-sulfonic

197

capacity (TEAC) assays. The TEAC assay measures the ability of compounds to

198

scavenge

199

(6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid; Sigma). The results were

200

expressed as TEAC in mmol of Trolox per kg on a dry-weight basis.43 All

To

the

measure

ABTS

antioxidant

radical

capacity,

acid)

cation

we

performed

(ABTS)/Trolox

(ABTS+)

in


2,2’-azino-bis

equivalent antioxidant

relation

to

Trolox

Page 11 of 36


201

experiments were carried out with three biological and three technical replicates.

202

Statistical analyses. Each value represents repeated independent experiments,

203

and the vertical bars expressed the arithmetic means ± standard deviations (SD).

204

Tukey’s test was used to calculate statistical significance, and the significant

205

differences between treatments and the untreated control are represented by ‘*’ at P

206

Development of Marker-Free Transgenic Potato Tubers Enriched in

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