Overexpression of HvHGGT Enhances Tocotrienol Levels and

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Overexpression of HvHGGT Enhances Tocotrienol Levels and Antioxidant Activity in Barley. Jianshu Chen, Cuicui Liu, Bo Shi, Yuqiong Chai, Ning Han, Muyuan Zhu, and HONGWU BIAN J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00439 • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 12, 2017

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Journal of Agricultural and Food Chemistry

Title:

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Overexpression of HvHGGT Enhances Tocotrienol Levels and Antioxidant Activity in Barley

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Authors:

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Jianshu Chen1, 2#, Cuicui Liu1 #, Bo Shi1, Yuqiong Chai1, Ning Han1, Muyuan Zhu1, Hongwu Bian1*

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1 Institute of Genetic and Regenerative Biology, Key Laboratory for Cell and Gene Engineering of Zhejiang

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Province, College of Life Sciences, Zhejiang University, Hangzhou, China, 310058;

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2 College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, China, 310014;

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# These authors contributed equally to this work.

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*Corresponding author:

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Dr H. W. Bian, Institute of Genetic and Regenerative Biology, Key Laboratory for Cell and Gene Engineering

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of Zhejiang Province, College of Life Sciences, Zhejiang University, Hangzhou, China;

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Email: [email protected]

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ABSTRACT

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Vitamin E is a potent lipid-soluble antioxidant and essential nutrient for human health. Tocotrienols are the

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major form of vitamin E in seeds of most monocots. It has been known that homogentisate geranylgeranyl

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transferase (HGGT) catalyzes the committed step of tocotrienol biosynthesis. In the present study, we

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generated transgenic barley overexpressing HvHGGT under endogenous D-Hordein promoter (proHor).

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Overexpression of HvHGGT increased seed size and seed weight in transgenic barley. Notably, total

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tocotrienol content increased by 10-15% in seeds of transgenic lines, due to the increased levels of δ-, β-

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and γ-tocotrienol, but not α-tocotrienol. Total tocopherol content decreased by 14-18% in transgenic lines,

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compared

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1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and lipid

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peroxidation assays. Compared to wild type, radical scavenging activity of seed extracts was enhanced by

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17-18% in transgenic lines. Meanwhile, the lipid peroxidation level was decreased by about 20% in

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transgenic barley seeds. Taken together, overexpression of HvHGGT enhanced the tocotrienol levels and

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antioxidant capacity in barley seeds.

to

wild

type.

The

antioxidant

activity

of

seeds

was

determined

by

using

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KEYWORDS:

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Tocotrienol; homogentisate geranylgeranyl transferase (HGGT); antioxidant activity; overexpression; barley

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INTRODUCTION

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Vitamin E, a lipophilic antioxidant, is an essential nutrient for human health, which consists of tocopherols

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and tocotrienols. These molecules contain a polar chromanol head group attached to a long isoprenoid side

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chain. The chromanol ring can be linked to a saturated phytyl side chain to form tocopherol, or to an

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unsaturated geranylgeranyl side chain to form tocotrienol. Based on the number and position of methyl

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groups on the chromanol head group, each of the two groups is composed of four alternative forms (α, β, γ

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and δ). Biological activity differs considerably among these eight isomers. α-Tocopherol has been the focus

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of vitamin E research and regarded as a major form with the most potent antioxidant and biological

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activity.1 In

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properties over tocopherols, including hypocholesterolemic, anti-cancer and neuroprotective activities

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which are not often exhibited by tocopherols.2-6 Owing to their health-promoting properties, tocotrienols

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are commercially produced as nutraceuticals mainly from rice bran and palm oil.

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Biosynthetic pathway for vitamin E has been elucidated several years ago (Figure 1).7 The committed step in

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tocotrienol biosynthesis is the condensation of homogentisate (HGA) with geranylgeranyl diphosphate

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(GGDP) to form 2-methyl-6-geranylgeranyl-1,4-benzoquinol (MGGBQ), which is catalyzed by homogentisate

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geranylgeranyl transferase (HGGT).8 In parallel to tocotrienol biosynthesis, tocopherols are synthesized by

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the

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2-methyl-6-phytyl-1,4-benzoquinol (MPBQ), which is catalyzed by homogentisate phytyltransferase (HPT).9

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Subsequent reactions in synthesis of both tocotrienol and tocopherol are ring methylations and ring

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cyclization, which are catalyzed by common enzymes, 2-methyl-6-phytylbenzoquinone methyltransferase

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(MPBQMT), tocopherol cyclase (TC) and γ-tocopherol methyltransferase (γ-TMT).10-13

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Up to date, HGGT homologue genes have only been identified in the monocot plants, such as barley, rice

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and wheat. It was reported that overexpression of barley HGGT (HvHGGT) in tobacco, Arabidopsis thaliana

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and maize resulted in a considerable ACS accumulation of total vitamin E, primarily as tocotrienols.8 While in Paragon Plus Environment

recent

condensation

years, tocotrienols have received great

of

homogentisate

with

3

phytyl

attentions due to its superior biological

diphosphate

(PDP)

to

form


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transgenic soybean expressing rice HGGT, there was only a slight accumulation of tocotrienols.14

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Considerable progress has been made in recent years to modify content and composition of tocopherol in

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vegetables and crops by overexpression of tocopherol biosynthesis genes. However, there are very few

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reports on metabolic engineering of tocotrienol biosynthesis in cereals.

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Barley is one of the world's major crops, mainly used for food, animal feed and malt. Moreover, barley is

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gaining renewed interest as a functional food due to its high content of bioactive compounds, including

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β-glucan, phenolics and vitamin E.15-17 Barley grains, as a good source of tocotrienols, contain a large

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amount of tocotrienols, at least above 70% of total vitamin E. The major vitamin E isomer in barley was

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α-tocotrienol, accounting for about 65% of total tocotrienols.15 Therefore, manipulation of the tocotrienol

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biosynthetic pathway in barley could further enhance the tocotrienol levels, and fortify the nutrition of grain.

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However, the improvement of micronutrients in barley has lagged behind the progress in other crops.18 One

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reason was the recalcitrance of barley to genetic transformation. Progress on barley transformation has

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been made in recent years. Our previous work demonstrated that overexpression of β-glucanase gene

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resulted in decreased β-glucan content and increased starch level in barley grains.19 It suggests that

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manipulation of a target gene can be an efficient approach to modify the nutrition content in barley.

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In the present study, we generated transgenic barley overexpressing HvHGGT under the control of the

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barley D-Hordein gene promoter (proHor). Vitamin E was analyzed by high-performance liquid

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chromatography (HPLC). Antioxidant activities were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH),

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2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and lipid peroxidation assays. We evaluated the

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effect of overexpression of HvHGGT on seed size, vitamin E levels, and antioxidant activity of barley seeds.

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MATERIALS AND METHODS

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Vector Construction

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Total RNA was extracted from the developing seedsPlus of barley (Hordeum vulgare L. cv Golden Promise) at 7 ACS Paragon Environment 4

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days after anthesis. Full-length of HvHGGT cDNA was amplified by reverse transcription polymerase chain

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reaction (RT-PCR). The primers used were as follow: F: 5' GGATCCGCGAGGATGCAAGCCGTCAC 3', R: 5'

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CTGCAGTAGTTCACATCTGCTGGCCCTT 3'. The amplified products were sequenced and then cloned into BamI

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and PstI sites downstream of the seed-specific promoter proHor in pCAMBIA13011.19 This construct was

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then transferred into Agrobacterium tumefaciens strain AGL1 with electroporation method (Eppendorf

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Electrporator 2510).

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Barley Transformation

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Barley transformation was performed by Agrobacterium tumefaciens-mediated co-cultivation approach

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described in our previous work.19 Plants of the spring barley cultivar Golden Promise were grown under

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natural conditions in experimental fields (Zhejiang University).

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PCR Detection and Southern Hybridization

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Transgenic plants were examined by PCR. Genomic DNA from transgenic barley leaf was extracted using the

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CTAB method. pCAMBIA13011 vector was served as positive control, whereas genomic DNA from wild-type

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barley was the negative control. For Southern blot analysis, total DNA was isolated from approximately 1 g

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of barley leaf. 30 μg of genomic DNA was digested with Hind III restriction enzyme (TaKaRa, Dalian, China) at

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37°C overnight, separated on 1% agarose gel and transferred to nylon membrane. Subsequent hybridization

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was performed using a digoxigenin-labeled Hpt-specific probe and Detection Starter Kit II (Roche Applied

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Science Com.) according to standard protocols.

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Real-Time Quantitative PCR and Semi-quantitative RT-PCR

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Total RNA was extracted from various barely tissues (leaf, root, immature embryo and mature seed) using

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TRIzol (Invitrogen, Carlsbad, CA, USA), and then treated with DNase I (Takara, Dalian, China). 1 μg RNA was

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used for First-strand cDNA synthesis using the PrimeScript RT reagent Kit (Takara, Dalian, China). Real-time

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quantitative PCR was performed using the Master cycler ep realplex system (Eppendorf, Hamburg, Germany)

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and the SYBR PrimeScript RT-PCR kit (Perfect Real Time; For semi-quantitative RT-PCR detection of ACS Paragon Plus TaKaRa). Environment 5


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HvHGGT expression, 3 µg of total RNA was used for reverse-transcription. HvACTIN was used as the internal

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control. The primers used for HvHGGT were as follow: F: 5' TTGCTTCTCTGCCGTCATAG 3', R: 5'

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GCTGTCAACAATATGCTTATGC 3'.

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Determination of Total Starch Content

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Barley seeds were ground to flour. Total starch contents were determined using the Total Starch Assay Kit

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(Megazyme International Ireland).

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Analysis of Vitamin E

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Vitamin E was extracted according to the previously described methods with some modifications.20 Seeds

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were ground to a fine powder in liquid nitrogen. 50 mg flour was extracted in 1mL methanol by vortex

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mixing vigorously for 10 min at room temperature. The samples were centrifuged at 10000 g for 5 min, and

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the supernatant was transferred to new tubs. The pellet was re-extracted twice. All the three supernatant

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were pooled, and dried under vacuum with a centrifugal evaporator (Labconco), then the residues were

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redissolved in 100 µL methanol/H2O (85:15, v / v), and filtered using a Millipore 0.45 µm membrane.

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Vitamin E were determined using an Agilent 1200 HPLC (Agilent Technologies)

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Phenomenex KinetexTM PFP column (2.6 μm, 150 × 4.6 mm; Phenomenex) and a fluorescence detector

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G1321A. The mobile phase used was methanol/H2O (85:15, v/v) with a flow rate of 1.0 mL/min. Sample

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components were detected by fluorescence with excitation at 298 nm and emission at 328 nm,

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respectively.21 Tocopherols and tocotrienols were quantified against external standard curves using

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authentic compounds (ChromaDex).

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Preparation of Samples

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100 mg barley flour was extracted with 1 mL 80% acetone (v/v) at room temperature for 4h using orbital

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shaker. The mixture was centrifuged at 10000 g for 10 min. The supernatant was used for the following

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measurement.

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Determination of Total Phenolic Content ACS Paragon Plus Environment 6

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with a

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Total phenolic content of the barley was determined according to the Folin-Ciocaletu method with some

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modifications.22 1mL of appropriately diluted extract was mixed with 2 ml of 10-fold diluted Folin–Ciocalteu

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reagent, and reacted for 5 min, then 2 mL of 7.5% Na2CO3 was added. After 1 h of incubation at room

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temperature in the dark, the absorbance was measured at 760 nm using a UV spectrophotometer. Total

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phenolic content were expressed as milligrams of gallic acid equivalents (GAE) per gram of barley flour.

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Determination of DPPH Radical-Scavenging Activity

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DPPH radical-scavenging activity was assayed according to previously described methods with some

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modifications.22 3mL of DPPH reagent (0.004% in methanol) was added to 50 µL of sample and mixed

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thoroughly. After incubation at room temperature in the dark for 1 h, the absorbance was measured at 517

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nm. Radical scavenging activity was calculated as ((Ac - At) / A c) ×100, where At and Ac represent absorbance

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readings with and without sample, respectively.

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Determination of Total Antioxidant Capacity

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Total antioxidant capacity assay is based on the abilities of antioxidants to scavenge the ABTS radical cation

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relative to a standard Trolox curve.22 The ABTS+ reagent was prepared by mixing 7 mM ABTS solution with

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2.45 mM potassium persulfate, and incubated at room temperature in the dark for 12–16 h. The ABTS+

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solution was diluted with 95% ethanol to an absorbance of approximately 0.70 at 734 nm prior to use. 3.0

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mL of ABTS+ solution was added to 20 µL of the sample and mixed vigorously. The reaction mixture was kept

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at room temperature in the dark for 6 min and the absorbance was measured at 734 nm immediately.

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Trolox with various concentrations in ethanol was prepared as a standard. The results were expressed in

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terms of Trolox equivalent antioxidant capacity (TEAC).

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Determination of Lipid Peroxidation

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Lipid peroxidation was determined by measuring malondialdehyde (MDA) content according to the method

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of Ho et al. with some modifications.14 Seeds were ground to a fine powder in liquid nitrogen. 500 mg flour

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was soaked in 5 mL of 0.1% trichloroacetic and Plus the homogenate ACSacid, Paragon Environment was centrifuged at 12000 g for 10 min. 7


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1 mL of supernatant was added to 4 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The

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mixture was heated at 95°C for 30 min and then cooled rapidly on ice. Following centrifugation at 12000 g

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for 10 min, the absorbance of the supernatant was measured at 532 nm and 600 nm. Malondialdehyde

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concentration was calculated using an extinction coefficient of 157 mM-1 cm-1.

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Statistical Analysis

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All experiments were repeated at least three times. The data shown represent mean ± standard deviation.

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Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Asterisks in the figures

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denote significant differences as follows: *P

Overexpression of HvHGGT Enhances Tocotrienol Levels and

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