Subscriber access provided by UNIV LAVAL
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
1
Journal of Agricultural and Food Chemistry
Title:
2 3
Overexpression of HvHGGT Enhances Tocotrienol Levels and Antioxidant Activity in Barley
4 5
Authors:
6
Jianshu Chen1, 2#, Cuicui Liu1 #, Bo Shi1, Yuqiong Chai1, Ning Han1, Muyuan Zhu1, Hongwu Bian1*
7
1 Institute of Genetic and Regenerative Biology, Key Laboratory for Cell and Gene Engineering of Zhejiang
8
Province, College of Life Sciences, Zhejiang University, Hangzhou, China, 310058;
9
2 College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, China, 310014;
10
# These authors contributed equally to this work.
11 12
*Corresponding author:
13
Dr H. W. Bian, Institute of Genetic and Regenerative Biology, Key Laboratory for Cell and Gene Engineering
14
of Zhejiang Province, College of Life Sciences, Zhejiang University, Hangzhou, China;
15
Email:
[email protected] ACS Paragon Plus Environment 1
Journal of Agricultural and Food Chemistry
Page 2 of 36
16
ABSTRACT
17
Vitamin E is a potent lipid-soluble antioxidant and essential nutrient for human health. Tocotrienols are the
18
major form of vitamin E in seeds of most monocots. It has been known that homogentisate geranylgeranyl
19
transferase (HGGT) catalyzes the committed step of tocotrienol biosynthesis. In the present study, we
20
generated transgenic barley overexpressing HvHGGT under endogenous D-Hordein promoter (proHor).
21
Overexpression of HvHGGT increased seed size and seed weight in transgenic barley. Notably, total
22
tocotrienol content increased by 10-15% in seeds of transgenic lines, due to the increased levels of δ-, β-
23
and γ-tocotrienol, but not α-tocotrienol. Total tocopherol content decreased by 14-18% in transgenic lines,
24
compared
25
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and lipid
26
peroxidation assays. Compared to wild type, radical scavenging activity of seed extracts was enhanced by
27
17-18% in transgenic lines. Meanwhile, the lipid peroxidation level was decreased by about 20% in
28
transgenic barley seeds. Taken together, overexpression of HvHGGT enhanced the tocotrienol levels and
29
antioxidant capacity in barley seeds.
to
wild
type.
The
antioxidant
activity
of
seeds
was
determined
by
using
30 31
KEYWORDS:
32
Tocotrienol; homogentisate geranylgeranyl transferase (HGGT); antioxidant activity; overexpression; barley
ACS Paragon Plus Environment 2
Page 3 of 36
Journal of Agricultural and Food Chemistry
33
INTRODUCTION
34
Vitamin E, a lipophilic antioxidant, is an essential nutrient for human health, which consists of tocopherols
35
and tocotrienols. These molecules contain a polar chromanol head group attached to a long isoprenoid side
36
chain. The chromanol ring can be linked to a saturated phytyl side chain to form tocopherol, or to an
37
unsaturated geranylgeranyl side chain to form tocotrienol. Based on the number and position of methyl
38
groups on the chromanol head group, each of the two groups is composed of four alternative forms (α, β, γ
39
and δ). Biological activity differs considerably among these eight isomers. α-Tocopherol has been the focus
40
of vitamin E research and regarded as a major form with the most potent antioxidant and biological
41
activity.1 In
42
properties over tocopherols, including hypocholesterolemic, anti-cancer and neuroprotective activities
43
which are not often exhibited by tocopherols.2-6 Owing to their health-promoting properties, tocotrienols
44
are commercially produced as nutraceuticals mainly from rice bran and palm oil.
45
Biosynthetic pathway for vitamin E has been elucidated several years ago (Figure 1).7 The committed step in
46
tocotrienol biosynthesis is the condensation of homogentisate (HGA) with geranylgeranyl diphosphate
47
(GGDP) to form 2-methyl-6-geranylgeranyl-1,4-benzoquinol (MGGBQ), which is catalyzed by homogentisate
48
geranylgeranyl transferase (HGGT).8 In parallel to tocotrienol biosynthesis, tocopherols are synthesized by
49
the
50
2-methyl-6-phytyl-1,4-benzoquinol (MPBQ), which is catalyzed by homogentisate phytyltransferase (HPT).9
51
Subsequent reactions in synthesis of both tocotrienol and tocopherol are ring methylations and ring
52
cyclization, which are catalyzed by common enzymes, 2-methyl-6-phytylbenzoquinone methyltransferase
53
(MPBQMT), tocopherol cyclase (TC) and γ-tocopherol methyltransferase (γ-TMT).10-13
54
Up to date, HGGT homologue genes have only been identified in the monocot plants, such as barley, rice
55
and wheat. It was reported that overexpression of barley HGGT (HvHGGT) in tobacco, Arabidopsis thaliana
56
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
Journal of Agricultural and Food Chemistry
Page 4 of 36
57
transgenic soybean expressing rice HGGT, there was only a slight accumulation of tocotrienols.14
58
Considerable progress has been made in recent years to modify content and composition of tocopherol in
59
vegetables and crops by overexpression of tocopherol biosynthesis genes. However, there are very few
60
reports on metabolic engineering of tocotrienol biosynthesis in cereals.
61
Barley is one of the world's major crops, mainly used for food, animal feed and malt. Moreover, barley is
62
gaining renewed interest as a functional food due to its high content of bioactive compounds, including
63
β-glucan, phenolics and vitamin E.15-17 Barley grains, as a good source of tocotrienols, contain a large
64
amount of tocotrienols, at least above 70% of total vitamin E. The major vitamin E isomer in barley was
65
α-tocotrienol, accounting for about 65% of total tocotrienols.15 Therefore, manipulation of the tocotrienol
66
biosynthetic pathway in barley could further enhance the tocotrienol levels, and fortify the nutrition of grain.
67
However, the improvement of micronutrients in barley has lagged behind the progress in other crops.18 One
68
reason was the recalcitrance of barley to genetic transformation. Progress on barley transformation has
69
been made in recent years. Our previous work demonstrated that overexpression of β-glucanase gene
70
resulted in decreased β-glucan content and increased starch level in barley grains.19 It suggests that
71
manipulation of a target gene can be an efficient approach to modify the nutrition content in barley.
72
In the present study, we generated transgenic barley overexpressing HvHGGT under the control of the
73
barley D-Hordein gene promoter (proHor). Vitamin E was analyzed by high-performance liquid
74
chromatography (HPLC). Antioxidant activities were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH),
75
2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and lipid peroxidation assays. We evaluated the
76
effect of overexpression of HvHGGT on seed size, vitamin E levels, and antioxidant activity of barley seeds.
77 78
MATERIALS AND METHODS
79
Vector Construction
80
Total RNA was extracted from the developing seedsPlus of barley (Hordeum vulgare L. cv Golden Promise) at 7 ACS Paragon Environment 4
Page 5 of 36
Journal of Agricultural and Food Chemistry
81
days after anthesis. Full-length of HvHGGT cDNA was amplified by reverse transcription polymerase chain
82
reaction (RT-PCR). The primers used were as follow: F: 5' GGATCCGCGAGGATGCAAGCCGTCAC 3', R: 5'
83
CTGCAGTAGTTCACATCTGCTGGCCCTT 3'. The amplified products were sequenced and then cloned into BamI
84
and PstI sites downstream of the seed-specific promoter proHor in pCAMBIA13011.19 This construct was
85
then transferred into Agrobacterium tumefaciens strain AGL1 with electroporation method (Eppendorf
86
Electrporator 2510).
87
Barley Transformation
88
Barley transformation was performed by Agrobacterium tumefaciens-mediated co-cultivation approach
89
described in our previous work.19 Plants of the spring barley cultivar Golden Promise were grown under
90
natural conditions in experimental fields (Zhejiang University).
91
PCR Detection and Southern Hybridization
92
Transgenic plants were examined by PCR. Genomic DNA from transgenic barley leaf was extracted using the
93
CTAB method. pCAMBIA13011 vector was served as positive control, whereas genomic DNA from wild-type
94
barley was the negative control. For Southern blot analysis, total DNA was isolated from approximately 1 g
95
of barley leaf. 30 μg of genomic DNA was digested with Hind III restriction enzyme (TaKaRa, Dalian, China) at
96
37°C overnight, separated on 1% agarose gel and transferred to nylon membrane. Subsequent hybridization
97
was performed using a digoxigenin-labeled Hpt-specific probe and Detection Starter Kit II (Roche Applied
98
Science Com.) according to standard protocols.
99
Real-Time Quantitative PCR and Semi-quantitative RT-PCR
100
Total RNA was extracted from various barely tissues (leaf, root, immature embryo and mature seed) using
101
TRIzol (Invitrogen, Carlsbad, CA, USA), and then treated with DNase I (Takara, Dalian, China). 1 μg RNA was
102
used for First-strand cDNA synthesis using the PrimeScript RT reagent Kit (Takara, Dalian, China). Real-time
103
quantitative PCR was performed using the Master cycler ep realplex system (Eppendorf, Hamburg, Germany)
104
and the SYBR PrimeScript RT-PCR kit (Perfect Real Time; For semi-quantitative RT-PCR detection of ACS Paragon Plus TaKaRa). Environment 5
Journal of Agricultural and Food Chemistry
Page 6 of 36
105
HvHGGT expression, 3 µg of total RNA was used for reverse-transcription. HvACTIN was used as the internal
106
control. The primers used for HvHGGT were as follow: F: 5' TTGCTTCTCTGCCGTCATAG 3', R: 5'
107
GCTGTCAACAATATGCTTATGC 3'.
108
Determination of Total Starch Content
109
Barley seeds were ground to flour. Total starch contents were determined using the Total Starch Assay Kit
110
(Megazyme International Ireland).
111
Analysis of Vitamin E
112
Vitamin E was extracted according to the previously described methods with some modifications.20 Seeds
113
were ground to a fine powder in liquid nitrogen. 50 mg flour was extracted in 1mL methanol by vortex
114
mixing vigorously for 10 min at room temperature. The samples were centrifuged at 10000 g for 5 min, and
115
the supernatant was transferred to new tubs. The pellet was re-extracted twice. All the three supernatant
116
were pooled, and dried under vacuum with a centrifugal evaporator (Labconco), then the residues were
117
redissolved in 100 µL methanol/H2O (85:15, v / v), and filtered using a Millipore 0.45 µm membrane.
118
Vitamin E were determined using an Agilent 1200 HPLC (Agilent Technologies)
119
Phenomenex KinetexTM PFP column (2.6 μm, 150 × 4.6 mm; Phenomenex) and a fluorescence detector
120
G1321A. The mobile phase used was methanol/H2O (85:15, v/v) with a flow rate of 1.0 mL/min. Sample
121
components were detected by fluorescence with excitation at 298 nm and emission at 328 nm,
122
respectively.21 Tocopherols and tocotrienols were quantified against external standard curves using
123
authentic compounds (ChromaDex).
124
Preparation of Samples
125
100 mg barley flour was extracted with 1 mL 80% acetone (v/v) at room temperature for 4h using orbital
126
shaker. The mixture was centrifuged at 10000 g for 10 min. The supernatant was used for the following
127
measurement.
128
Determination of Total Phenolic Content ACS Paragon Plus Environment 6
equipped
with a
Page 7 of 36
Journal of Agricultural and Food Chemistry
129
Total phenolic content of the barley was determined according to the Folin-Ciocaletu method with some
130
modifications.22 1mL of appropriately diluted extract was mixed with 2 ml of 10-fold diluted Folin–Ciocalteu
131
reagent, and reacted for 5 min, then 2 mL of 7.5% Na2CO3 was added. After 1 h of incubation at room
132
temperature in the dark, the absorbance was measured at 760 nm using a UV spectrophotometer. Total
133
phenolic content were expressed as milligrams of gallic acid equivalents (GAE) per gram of barley flour.
134
Determination of DPPH Radical-Scavenging Activity
135
DPPH radical-scavenging activity was assayed according to previously described methods with some
136
modifications.22 3mL of DPPH reagent (0.004% in methanol) was added to 50 µL of sample and mixed
137
thoroughly. After incubation at room temperature in the dark for 1 h, the absorbance was measured at 517
138
nm. Radical scavenging activity was calculated as ((Ac - At) / A c) ×100, where At and Ac represent absorbance
139
readings with and without sample, respectively.
140
Determination of Total Antioxidant Capacity
141
Total antioxidant capacity assay is based on the abilities of antioxidants to scavenge the ABTS radical cation
142
relative to a standard Trolox curve.22 The ABTS+ reagent was prepared by mixing 7 mM ABTS solution with
143
2.45 mM potassium persulfate, and incubated at room temperature in the dark for 12–16 h. The ABTS+
144
solution was diluted with 95% ethanol to an absorbance of approximately 0.70 at 734 nm prior to use. 3.0
145
mL of ABTS+ solution was added to 20 µL of the sample and mixed vigorously. The reaction mixture was kept
146
at room temperature in the dark for 6 min and the absorbance was measured at 734 nm immediately.
147
Trolox with various concentrations in ethanol was prepared as a standard. The results were expressed in
148
terms of Trolox equivalent antioxidant capacity (TEAC).
149
Determination of Lipid Peroxidation
150
Lipid peroxidation was determined by measuring malondialdehyde (MDA) content according to the method
151
of Ho et al. with some modifications.14 Seeds were ground to a fine powder in liquid nitrogen. 500 mg flour
152
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
Journal of Agricultural and Food Chemistry
Page 8 of 36
153
1 mL of supernatant was added to 4 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid. The
154
mixture was heated at 95°C for 30 min and then cooled rapidly on ice. Following centrifugation at 12000 g
155
for 10 min, the absorbance of the supernatant was measured at 532 nm and 600 nm. Malondialdehyde
156
concentration was calculated using an extinction coefficient of 157 mM-1 cm-1.
157
Statistical Analysis
158
All experiments were repeated at least three times. The data shown represent mean ± standard deviation.
159
Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Asterisks in the figures
160
denote significant differences as follows: *P