This study genetically engineered the tocopherol biosynthetic pathway in Arabidopsis thaliana by overexpressing five genes (HPPD, VTE2, VTE3, VTE1, and VTE4) involved in tocopherol production, both individually and in combinations. The results showed that elevated expression of these biosynthetic genes affected total tocopherol content and composition. Additionally, engineering the tocopherol pathway also impacted endogenous ascorbate and glutathione pools in the leaves. Further analysis found that genes in the Halliwell-Asada antioxidant cycle were upregulated. These findings provide insight into the relationship between lipid-soluble vitamin E and water-soluble antioxidants vitamin C and

1. Plant Science 178 (2010) 312–320
Contents lists available at ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Engineering tocopherol biosynthetic pathway in Arabidopsis leaves and its
effect on antioxidant metabolism
Yin Li a, Yin Zhou a, Zinan Wang a, Xiaofen Sun a, Kexuan Tang a,b,*
a
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai 200433, PR China
b
Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,
Shanghai Jiao Tong University, Shanghai 200030, PR China
A R T I C L E I N F O A B S T R A C T
Article history: With genetic manipulation, five genes (HPPD, VTE2, VTE3, VTE1, and VTE4), which encode enzymes
Received 24 November 2009 involved in tocopherol biosynthesis, were over-expressed in model plant Arabidopsis thaliana, either
Received in revised form 17 January 2010 alone or in couple combinations (VTE2 + VTE4 and VTE3 + VTE4), to value and compare the roles of
Accepted 19 January 2010
enzymes played in tocopherol biosynthetic pathway under the same genetic background. Our results
Available online 28 January 2010
suggested that, elevated expression level of biosynthetic pathway gene affected either total tocopherol
content or composition, it is recommended to choose two or more enzymes with different functions for
Keywords:
genetic manipulation. It was also found that metabolic engineering of tocopherol biosynthetic pathway
Arabidopsis thaliana
Biosynthetic pathway
affected endogenous ascorbate and glutathione pools in leaves. Further study suggested that expression
Halliwell–Asada cycle levels of genes encoding enzymes of Halliwell–Asada cycle were up-regulated, such as APX, DHAR and
Over-expression MDAR. These findings provide hints on the relationship of lipid-soluble antioxidant vitamin E and water-
Tocopherol soluble antioxidants vitamin C and glutathione, which will help to perfect theory in plant physiology and
Vitamin E give practical instruction for metabolic engineering.
ß 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction adults (http://ods.od.nih.gov/factsheets/vitamine.asp). Higher dai-
ly vitamin E doses have been needed for cancer reduction, immune
Vitamin E is an essential nutrient of daily diet for humans and response, and cardiovascular benefits [2]. Among the family, a-
animals. Naturally occurring vitamin E exists in eight chemical tocopherol is believed to have the highest vitamin E activity to
forms (a-, b-, g-, and d-tocopherol and a-, b-, g-, and d- meet human requirements, and it is preferentially retained and
tocotrienol) that have varying levels of biological activity. The distributed throughout the body [3]. Naturally synthesized a-
nutritional values of vitamin E were affirmed in 1922 [1]. The tocopherol, which is a single (R, R, R) stereoisomer, has more
National Institutes of Health (NIH) currently suggests a recom- activity than chemically synthesized a-tocopherol [4].
mended daily allowance (RDA) of 15–19 mg a-tocopherol for Tocopherols can be synthesized only in photoautotrophy
organisms, including plants and other oxygenic, photosynthetic
organisms. Although the tocopherol biosynthetic pathway had
Abbreviations: APX, ascorbic acid peroxidase; AsA, ascorbic acid (vitamin C); DHA, been elucidated from 1979 [5], the genetic analysis of the pathway
dehydroascorbic acid; DHAR, dehydroascorbic acid reductase; GDPME, GDP-D- and key enzymes had only commenced since 1990s, with the
mannose-3, 5-epimerase; GDPMPPase, GDP-D-mannose pyrophosphorylase; GSH,
approaches of genetic and genomics-based methodologies in the
glutathione; GSSG, oxidized glutathione; HGA, homogentisic acid; HPLC, high
performance liquid chromatography; HPPD, p-hydroxyphenylpyruvic acid dioxy- model organisms Arabidopsis thaliana and Synechocystis sp.
genase; L-GalDH, L-galactose dehydrogenase; L-GalPPase, L-galactose 1-P phospha- PCC6803.
tase; L-GLDH, L-galactono-g-lactone dehydrogenase; MDAR, monodehydroascorbic Tocopherol biosynthesis mainly takes place in plastids of higher
acid; MEP, methylerythritol phosphate; NC, non-transgenic control; PDP, phytyl-
plants. The tocopherol biosynthetic pathway utilizes two com-
diphosphate; PQ8, plastochromanol-8; PUFAs, protect polyunsaturated fatty acid
chains; RDA, recommended daily allowance; ROS, reactive oxygen species; VTC2,
pounds from different metabolic pathways as precursors, which
GDP-L-galactose phosphorylase; VTE1, tocopherol cyclase; VTE2, homogentisate include homogentisic acid (HGA), derived from cytosolic shikimate
phytyltransferase; VTE3, 2-methyl-6-phytylplastoquinol methyltransferases; metabolic pathway for head group and phytyldiphosphate (PDP)
VTE4, g-tocopherol methyltransferase. [6] from the plastidic methylerythritol phosphate (MEP) pathway
* Corresponding author at: State Key Laboratory of Genetic Engineering, School of
for tail synthesis [7,8] (Fig. 1). There are at least five enzymes
Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan
University, 220 Handan Road, Shanghai 200433, PR China. involved in the biosynthesis of tocopherols, excluding the bypass
E-mail addresses: kxtang1@yahoo.com, kxtang@fudan.edu.cn (K. Tang). pathway of phytyl-tail synthesis and utilization (Table 1). HGA is
0168-9452/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2010.01.004

2. Y. Li et al. / Plant Science 178 (2010) 312–320 313
this work, genes encoding these enzymes were cloned from the
model plant A. thaliana and constitutively over-expressed, alone or
in combination, in order to achieve this assessment.
In metabolic engineering, when target nutritional product is
increasing, other related nutritional products in the same
bioreactor might be affected, in some cases they would decrease.
Many studies reported the antioxidant properties of tocopherols,
such as photo protection and reduction of lipid peroxidation by
reducing lipid peroxyl radicals to their corresponding hydroper-
oxides [16,17,18]. As lipid-soluble antioxidant, tocopherols locate
mainly on membranes of many cellular compartments, and will be
oxidized to tocopherol radical [19]. There are two important
water-soluble antioxidants in plant cells—ascorbic acid (vitamin C,
AsA) and glutathione (GSH), and they can scavenge reactive oxygen
species by Halliwell–Asada cycle during normal metabolism and
particularly during stress [20]. Especially vitamin C is not only an
important antioxidant in plant physiology, but also a metabolic
Fig. 1. Simplified tocopherol biosynthetic pathway from shikimate and MEP product with important nutritional and physiological values for
pathways. humans and animals. Former works indicated there might be
relationship existing among tocopherol, AsA and glutathione
produced from the tyrosine aromatic amino acid catabolite p- contents [21,22]. It was reported [22] that deficiency in one
hydroxyphenylpyruvate (HPP) by the cytosolic enzyme 4-hydro- antioxidant in tocopherol, AsA or glutathione led to increased
xyphenylpyruvate dioxygenase (HPPD) [9]. Condensation of HGA oxidative stress and the concomitant increase in alternative
and PDP is catalyzed by homogentisate phytyltransferases (VTE2) antioxidants. In some other cases, such as sunflower cell lines,
[10]. The product of this reaction, 2-methyl-6-phytylbenzoquinol high content of tocopherols leads to higher content levels of
(MPBQ), is the first phytylquinol intermediate in the pathway and ascorbate and glutathione pools [21]. However, in the study which
can be methylated to 2,3-dimethyl-6-phytyl-1, 4-benzoquinol observed the plant responses to oxidant stress in the presence of
(DMPBQ) by MPBQ methyltransferase (VTE3) [11]. Both MPBQ and Cu or Cd [23], the correlation between high tocopherol and low
DMPBQ are substrates for tocopherol cyclase (VTE1) to yield the ascorbate/glutathione levels was not seen.
first tocopherols of the pathway, d-tocopherol and g-tocopherol, In this study AsA and glutathione contents in transgenic lines
respectively [12]. Both d- and g-tocopherol can be methylated by over-expressing tocopherol biosynthetic pathway genes were
g-tocopherol methyltransferase (VTE4) to yield b- and a- analyzed in order to assess whether accumulation of vitamin E
tocopherol, respectively [13]. affected other antioxidants in plant. Furthermore, some studies
As a member of plant secondary metabolites, vitamin E has were done in order to explain the change of vitamin C and
various biological and pharmaceutical functions to humans as well glutathione in transgenic lines.
as to plants. Crops and vegetables are the best source for natural
vitamin E. Nevertheless, vitamin E is of low content, and the 2. Materials and methods
composition of the eight forms needs to optimize. Recently,
metabolic engineering has been widely applied in order to achieve 2.1. Plant materials and growth conditions
higher yields of specific metabolites. The in-depth understanding
of biosynthetic pathways, along with the increasing number of Seeds of wild-type Arabidopsis (Columbia ecotype) were
cloned genes involved in biosynthesis, enable the exploration of sterilized with chlorox and spread on Murashige and Skoog
metabolic engineering as a potential effective approach to increase (1962) plates, then treated at 4 8C for 3–5 days and induced for
the yield of specific metabolites by enhancing rate-limiting steps germination in 24 h-white-light for 6 days. Then the seedlings
or by blocking competitive pathways [14]. Increasing vitamin E were transferred into soil at 20 8C under 16-h photoperiod of light
content and a-tocopherol composition in vegetables and crops has at 120 mmol mÀ2 sÀ1.
been an important aim for metabolic engineering. Strategies as to
over-expressing different genes involved in the pathway have been 2.2. cDNA generation and vector construction
employed. Although significant work has been done [15], it is still
hard to assess the relative importance of each enzyme in For construction of plant expression vectors, myc tag was used
tocopherol biosynthetic pathway, due to different genetic back- as screening labels and sub-cloned into XhoI and PstI sites of
ground and various manipulation used in former studies. Valuation pBluescript SK+ vector (pBS; Stratagene) to form the vector
of the contribution of different enzymes under the same genetic pBSmyc (a gift from Prof. Hongquan Yang, SIPPE, CAS). Total RNA
background will be essential to provide effective strategies for was isolated from leaves of Arabidopsis thaliana (Columbia
large-scale commercial production of biosynthetic tocopherol. In ecotype) by using TRIzol reagent (GIBCO/BRL). The cDNAs of
Table 1
Enzymes, basic functions, loci and genes encoding tocopherol biosynthetic enzymes in Arabidopsis thaliana.
Pathway enzyme Basic function Arabidopsis
Gene Locus
p-Hydroxyphenylpyruvic acid dioxygenase (HPPD) Head group synthesis PDS1 At1g06570
Homogentisate prenyltransferase (HPT) Prenylation of HGA VTE2 At2g18950
2-Methyl-6-phytylbenzoquinone methyltransferase (MPBQ MT) Methylation of MPBQ and MGGBQ VTE3 At3g63410
Tocopherol cyclase (TC) Cyclization VTE1 At4g32770
g-Tocopherol methyltransferase (g-TMT) Methylation of d- and g-tocopherol VTE4 At1g64970

3. 314 Y. Li et al. / Plant Science 178 (2010) 312–320
Fig. 2. Schematic representation of the transformation vector construct used in this study. (A) Schematic representation of the single-gene transformation vector. (B)
Schematic representation of dual-gene transformation vector. LB, left border; RB, right border; 35S, cauliflower mosaic virus 35S promoter; 35S polyA, cauliflower mosaic
virus 35S polyA terminator; Nos, nopaline synthase gene terminator.
At-HPPD, VTE2, VTE3, VTE1, and VTE4 (Genbank accession Nos. screened on Murashige–Skoog (MS) basal medium supplemented
AY072329, AY089963, AB054257, NM119430 and AF104220) were with 50 mg mLÀ1 hygromycin. More than fifty independent lines
prepared by using TaKaRa One Step RT-PCR kit (Code No.: expressing transgene were transferred to soil and screened with 1%
DRR024A, TaKaRa, Japan). The amplified cDNA fragments of At- herbicide (glufosinate-ammonium). Screening of T1 and T2 seeds
HPPD, VTE2, VTE3, and VTE1 with an introduced HindIII site at the were performed with herbicide in order to obtain transgenic lines
start codon (ATG) and an introduced BamHI site before the stop which accorded with Mendel’s Law. Polymerase chain reaction
codon were digested, and sub-cloned into HindIII–BamHI site of (PCR) and protein gel blot analysis were also performed on
vector pBSmyc to obtain the expression cassette with recombina- independent transgenic lines (random chosen) of each generation,
tion sequence of target gene and myc. The amplified fragment of in order to further confirm and check the homozygosis and genetic
VTE4 with an introduced XhoI site at the start codon and an stability. Homozygous seeds could be obtained in T4 generation.
introduced BamHI site before the stop codon was digested, and The screening of co-expression transgenic plants was performed
sub-cloned into XhoI–BamHI site of pBSmyc. The reading frame of with PCR and western blot analysis, while herbicide could not be
pBS-target gene–myc was confirmed to be correct by sequencing used because anti-herbicide gene was not contained in the binary
from both strands. The recombinant sequences of target gene–myc vectors.
were introduced into pET28a vector and over-expressed in E. coli in
order to detect biological activity of fusing proteins in vitro. The 2.4. Molecular analysis of transgenic lines
recombinant sequences of target gene–myc were sub-cloned into
the botany expression vector PHB (a gift from Prof. Hongquan Genomic DNA was isolated from the transgenic plants and NC
Yang, SIPPE, CAS) [24], located downstream of double 35S CaMV plant with a CTAB method [26]. The presence of the transgene was
promoters and upstream of rbcS polyA terminator (Fig. 2A). detected by PCR.
In order to explore the function of two enzymes in plant Protein gel blot analysis was performed as described previously
tocopherol biosynthetic pathway, three of them were chosen for with minor modifications [27]. Fifty mg of total protein, deter-
dual expression vector construction. Two co-expression combina- mined by using the DC Protein Assay Kit (Bio-Rad, Hercules, CA),
tions were designed: VTE2 + VTE4 and VTE3 + VTE4. The target was fractionated on 12% SDS-PAGE mini-gel and blotted onto a
genes were also recombinant with myc. In order to put two genes in nitrocellulose membrane (PerkinElmer, 0.45 mm). The blots were
one vector, the expression cassette of vector pBI121 (Clontech) was probed with the primary antibody c-Myc (9E10) (sc-40, mouse
digested with HindIII and EcoRI, and then sub-cloned into the monoclonal IgG1, Santa Cruz Biotechnology) [diluted in PBST
HindIII–EcoRI site of pCAMBIA1304 vector to construct the co- (80 mmol LÀ1 Na2HPO4, 20 mmol LÀ1 NaH2PO4, 100 mmol LÀ1
expression vector. VTE4–myc fraction was sub-cloned into XbaI– NaCl, and 0.1% Tween 20)], washed with PBST three times, reacted
SacI site, while VTE2–myc and VTE3–myc fragment were cloned into with goat anti-mouse IgG-AP (sc-2047, Santa Cruz Biotechnology),
BglII–BstEII site, respectively (Fig. 2B). washed, and exposed with alkali phosphatase for 3 min.
[The primers used for cloning were listed in Supplement Table
2]. 2.5. Real-time PCR
2.3. Transformation and screening of transgenic plants Total RNA was isolated and genomic DNA was removed by
treatment with RQ1-RNase free DNase (Promega, Madison, WI).
The binary vector PHB contains hygromycin resistant gene and One microgram of total RNA was reverse transcribed to generate
anti-herbicide gene (BAR) inside the T-DNA for the selection of cDNA in 20 mL reactions for each sample using a Toyobo Rever Tra-
transformants. The PHB:target gene constructs were transformed Plus-Kit according to manufacturer’s recommendations (Toyobo,
into Agrobacterium tumefaciens C58C1 (pGV3101; rifampicin Osaka, Japan). An aliquot of cDNA corresponding to 10 pg – 10 ng
resistance) and the floral-dip method was used for Arabidopsis total RNA was used in each real-time PCR assay (SYBR1 ExScript
transformation [25]. The plants harboring empty plant expression RT-PCR Kit, TaKaRa, Shiga, Japan). Ubiquitin RNA was used to
vector were used as non-transgenic control (NC). T0 seeds were normalize RNA concentrations. Standard curves were constructed

4. Y. Li et al. / Plant Science 178 (2010) 312–320 315
for each gene and were used to calculate the corresponding mRNA
concentrations.
[The primers used in the real-time PCR assays were listed in
Supplement Table 3].
2.6. Analyses of vitamin E, vitamin C, and glutathione
Fifty independent plants (T4 generation) were chosen from the
same transgene line for vitamin E, vitamin C, and glutathione
analyses. Data of one transgene line were collected for statistics.
For each plant, the leaves of 3–4 weeks were harvested. Freeze-
dried material (150 mg) was ground in liquid nitrogen, and then
extracted with 4 mL of n-hexane in dim light and in the presence of
argon to prevent the oxidation of vitamin E. After centrifugation at
4000 Â g for 10 min, the clear supernatant was taken and the pellet
was re-extracted twice with 2 mL n-hexane. All the resulting
Fig. 3. Phenotype of Arabidopsis transgenic lines. (A–C) Transgenic seedlings
supernatants were pooled, evaporated to dryness under nitrogen,
transferred in soil for 3 days, 10 days, and 17 days. (D and E) Transgenic lines and
dissolved in 750 mL of methanol, and stored at À80 8C until being non-transgenic control in full blooms. There are no significant differences between
analyzed. transgenic lines and NC.
The methanol extracts were resolved on a Phenomenex C18
reverse-phase column (Calesil ODS-100, 5 mm, 4.6 mm
I.D. Â 250 mm length) at 30 8C with a methanol:isopropanol not be detected (Fig. 4C). PCR and western blot could help to
(95:5, v/v) mobile phase for 30 min at the flow rate of 1 mL minÀ1 confirm the plants harboring target transgene for each transfor-
1 to equilibrate the column. Vitamin E was detected by mation event.
fluorescence with excitation at 292 nm and emission at 325 nm.
Tocopherols and tocotrienols were identified by retention time and 3.2. Transcript changes of tocopherol biosynthetic pathway genes in
quantified relative to dilution series of standards. Quantification of transgenic lines
vitamin E was carried out by measuring peak areas using
chromatograph data system D-2000 Elite software (Hitachi, Japan). To access gene expression levels of transgenic plants, mRNA
Analyses of vitamin C were performed based on the method levels of tocopherol biosynthetic pathway genes were measured by
described by Kato and Esaka [28]. The vitamin C was analyzed in
leaves derived from the same plant which had tocopherol analyses
performed. Total ascorbate pool (AsA + DHA) was measured by
reducing DHA to AsA with dithiothreitol and the reductive ratio
calculated as [AsA/(AsA + DHA)] Â 100. HPLC analysis of AsA was
performed using a C18 reversed-phase column (Calesil ODS-100,
5 mm, 4.6 mm I.D. Â 250 mm length) at 30 8C with a 0.1% oxalic
acid:methanol (95:5, v/v) mobile phase for 30 min at the flow rate
of 1 mL minÀ1 to equilibrate the column. Samples (20 mL) were
injected and analyzed with an UV detector at 254 nm. AsA were
identified by retention time and quantified relative to dilution
series of AsA standards.
Glutathione was detected as described by Griffith [29].
Glutathione was extracted from frozen Arabidopsis leaves with
6% (v/v) TCA. The extract was neutralized to pH 8.0 with 1 mol LÀ1
K2CO3. Oxidized glutathione (GSSG) was reduced to 2Â GSH with
dithiothreitol. Total glutathione was determined using a cycling
assay based on the reaction with 2-nitrobenzoic acid and detected
by fluorescence with excitation at 380 nm and emission at 470 nm.
The reductive ratio calculated as [GSH/(GSH + GSSG)] Â 100.
3. Results
3.1. Arabidopsis transformation and molecular analysis of transgenic
plants
Different constructs with single (HPPD, VTE2, VTE3, VTE1, and
VTE4) and dual (VTE2 + VTE4 and VTE3 + VTE4) genes were
Fig. 4. Molecular detection of transgenic lines. (A) PCR analyses for the presence of
introduced into Arabidopsis. There were no developmental
myc and hygromycin (hyg) fragments in single transgenic lines. M, DL-2000 Marker
phenotypes in transgenic lines (Fig. 3). The hygromycin and (100–2000 bp); PC, vector used for transgenic (positive control); NC, non-
herbicide resistant plants of single-gene transgenic lines were transgenic control; HPPD, VTE2, VTE3, VTE1, and VTE4 showed representative
screened by PCR with hygromycin and myc gene primers (Fig. 4A). plants of corresponding transgenic lines. (B) PCR analyses for the presence of
For dual-gene transgenic lines, PCR was performed to analyze the transgenes in dual transgenic lines. VTE2 + VTE4 and VTE3 + VTE4 showed
representative plants of corresponding dual transgenic lines. (C) Western blots
presence of the transgene (Fig. 4B). The transgenic lines over- of transgenic lines. HPPD, VTE2, VTE3, VTE1, and VTE4 showed fusing proteins with
expressing target protein could be screened by specific myc-tag myc tag. VTE2 + VTE4, VTE2–myc and VTE4–myc fusing proteins; VTE3 + VTE4,
antibody, with which the corresponding endogenous protein could VTE3–myc and VTE4–myc fusing proteins.

5. 316 Y. Li et al. / Plant Science 178 (2010) 312–320
Fig. 5. Gene expression level of tocopherol biosynthetic pathway key enzymes in
transgenic plant leaves. Y axis, relative mRNA expression; NC, non-transgenic
control; HPPD, VTE2, VTE3, VTE1, and VTE4 showed representative plants of
corresponding transgenic lines; VTE2 + VTE4 and VTE3 + VTE4 showed
representative plants of corresponding dual transgenic lines.
real-time PCR in NC and transgenic lines. The expression levels of
target gene were elevated in both single- and dual-gene transgenic
lines (Fig. 5), suggesting that exogenous induction elevated
expression level of target gene in transgene lines. Further study
showed that the changes of target gene over-expression had
limited effect on the transcription of other tocopherol biosynthetic
pathway genes in transgenic lines (P > 0.05).
3.3. Genetic engineering of tocopherol biosynthetic pathway results in
accumulation of vitamin E and shift in a-tocopherol composition in
leaves
To assess the contributions of different enzymes in tocopherol
biosynthetic pathway, total vitamin E content and a-tocopherol
composition were analyzed. Total vitamin E content was
(14.28 Æ 2.26) pmol mgÀ1(FW) (n = 50, ÆSD, same below) and a-
tocopherol was about (87.5 Æ 7.1) % of all vitamin E forms in leaves of
NC. In this study, homologous over-expression of HPPD could increase
at least 43% total vitamin E content in leaves with a-tocopherol
composition increased modestly (88.1 Æ 7.5)%. However, accumulated
tocotrienols were not observed in this study. Over-expression of VTE2
would increase total vitamin E in Arabidopsis leaves. Among fifty
independent transgenic lines, total vitamin E increased to
(29.71 Æ 4.31) pmol mgÀ1(FW) in average, and the best produced
44.71 pmol mgÀ1(FW) vitamin E with only 85.7% a-tocopherol
composition. The function of over-expression of VTE3 did not have
distinct function in vitamin E content (14.68 Æ 1.64) pmol mgÀ1 (FW),
while a-tocopherol composition was elevated to 91.8% in the best
transgenic line. Over-expression of VTE1 or VTE4 alone significantly
affected tocopherol composition. Over-expression of VTE1 mainly
increased the proportion of g-tocopherol and its isoforms, while a- Fig. 6. Average vitamin E, vitamin C and glutathione contents in leaves of
tocopherol content did not show remarkable increase compared with representative transgenic Arabidopsis lines. (A) Vitamin E contents and a-
tocopherol composition of transgenic lines and NC, measured in pmol mgÀ1
NC. In transgenic line which expressed highest level of VTE1, the a-
(FW). The numbers above the bars showed a-tocopherol composition, calculated by
tocopherol only possessed 59.2% in total vitamin E, and the total [a-tocopherol/(a-tocopherol + g-tocopherol)] Â 100. (B) Dehydroascorbate (DHA)
vitamin E increased to (24.51 Æ 2.69) pmol mgÀ1 (FW). Over-expres- and ascorbic acid (AsA) contents in the same NC and transgenic lines, measured in
sion of VTE4 elevated both content and composition [(96.8 Æ 3.1) % in mmol gÀ1 (FW). The numbers above the bars showed the reductive ratio of vitamin
average] of a-tocopherol in Arabidopsis leaves, and increased the total C, calculated by [AsA/(AsA + DHA)] Â 100. (C) Glutathione (GSH) contents in the
same NC and transgenic lines, measured in mmol gÀ1 (FW). The numbers above the
vitamin E content to (16.96 Æ 2.87) pmol mgÀ1 (FW) (average of 50 bars showed the reductive ratio of reduced glutathione, calculated by [GSH/
independent transgenic lines). The best transgenic line had total (GSH + GSSG)] Â 100.
vitamin E content to (18.36 Æ 0.95) pmol mgÀ1 (FW), and a-tocopherol
composition was 97.1 (Fig. 6A).

6. Y. Li et al. / Plant Science 178 (2010) 312–320 317
The dual-gene transgenic lines showed multiple functions of
both enzymes. The VTE2 + VTE4 dual transgenic lines increased
total vitamin E content to (64.55 Æ 3.21) pmol mgÀ1 (FW) for the
best performing events, which was higher than VTE2 or VTE4 single
over-expression lines. In addition, the VTE3 + VTE4 dual transgenic
line had a-tocopherol composition increased to 97.9% in the best
transgenic line by the expense of other tocopherol forms, and the total
vitamin E content was about (16.89 Æ 1.58) pmol mgÀ1 (FW)
(Fig. 6A).
3.4. Tocopherol accumulation may affect ascorbate pool and its
reductive ratio in Arabidopsis leaves
To assess whether elevated vitamin E had influence on vitamin
C, bioactive compounds in leaves were measured in both NC and
single-gene over-expressing lines, including total vitamin E
content, a-tocopherol composition, vitamin C content, and
reductive ratio of AsA.
As shown in Fig. 6B, the endogenous ascorbate pool was
observed decrease in the vitamin E up-regulated plants, especially
in the a-tocopherol increased lines. AsA content in HPPD over-
expressing lines [(2.98 Æ 0.20) mmol LÀ1 (FW)] did not have obvious
difference with non-transgenic line [(3.07 Æ 0.19) mmol LÀ1 (FW)].
AsA content decreased to (0.94 Æ 0.08) mmol LÀ1 (FW) in the best
VTE2 over-expressing line, whose vitamin E level increased by 3.7-
fold compared with the NC. In dual transgenic line VTE2 + VTE4, which
had remarkable increase in total vitamin E content (4.5-fold), vitamin
C was observed decrease to (0.84 Æ 0.07) mmol LÀ1 (FW). It was also
found that the level of AsA changed in VTE3, VTE1, and VTE4 over-
expressing lines and VTE3 + VTE4 dual transgenic line, which had a-
tocopherol composition changed. For VTE3 lines, AsA decreased to
(1.65 Æ 0.13) mmol LÀ1 (FW). VTE1 and VTE4 over-expressing lines
showed similar decreasing trends in AsA, which had decreased to
(0.90 Æ 0.13) mmol LÀ1 (FW) and (0.66 Æ 0.10) mmol LÀ1 (FW), re- Fig. 7. Gene expression level of biosynthetic pathway gene and vitamin C content.
VTE1 expression lines were taken for example. (A) VTE1 expression level in VTE1
spectively. In VTE3 + VTE4 dual transgenic line, vitamin C was
transgenic lines. (B) Vitamin C content in corresponding transgenic lines.
observed decrease to (0.65 Æ 0.10) mmol LÀ1 (FW). Our data pre-
sented here indicated that, ascorbate pool might be influenced by the
change in vitamin E pool (Fig. 6B). Correlation analyses were showed C content. In HPPD transgenic line, the glutathione content was
negative correlation between vitamin E content and AsA content (0.60 Æ 0.03) mmol LÀ1 (FW), which showed no obvious difference
(r = À0.38, P = 0.0053, n = 50). between NC line [(0.63 Æ 0.04) mmol LÀ1 (FW)]. The VTE3 transgenic
The reductive ratio of AsA was also observed in this study line had modest decrease in glutathione content
(Fig. 6B). In the single transgenic lines VTE2, VTE1, and VTE4, whose [(0.44 Æ 0.03) mmol LÀ1 (FW)]. In the VTE2, VTE1, and VTE4 transgenic
AsA level strongly decreased, the reductive ratio of AsA greatly lines, the glutathione contents were (0.31 Æ 0.03) mmol LÀ1 (FW),
increased (87.3%, 90.7%, 91.9%, and 95.5% for NC, VTE2, VTE1, and (0.34 Æ 0.02) mmol LÀ1 (FW), and (0.28 Æ 0.02) mmol LÀ1 (FW), re-
VTE4). The increase in composition of reductive ratio was also spectively. In dual transgenic lines VTE2 + VTE4 and VTE3 + VTE4,
obvious in the transgenic lines VTE3 90.8%. On the contrary, no there were also existing similar trends, and the glutathione contents
significant change in reductive ratio was observed in HPPD over- were (0.25 Æ 0.02) mmol LÀ1 (FW) and (0.24 Æ 0.03) mmol LÀ1 (FW)
expressing lines 87.6%. In dual transgenic lines VTE2 + VTE4 and (Fig. 6C). Correlation analyses were showed negative correlation
VTE3 + VTE4, the reductive ratio of AsA increased to 93.3% and between vitamin E content and glutathione content (r = À0.46,
93.5%, respectively. P = 0.0044, n = 50). The content of AsA and glutathione had positive
It was also observed that the lines which had high expression correlation (r = 0.98, P = 0.020, n = 50).
level of tocopherol biosynthesis gene would show low AsA content. Considering the reductive glutathione ratio, it seemed that all
Take VTE1 transgene line for example. It seemed that the lines with transgenic lines had increase compared with NC (70.7%). For
high VTE1 expression level (Fig. 7A, also with high total tocopherol single-gene transgenic line, the ratio was 73.2%, 76.3%, 72.5%,
content) would have low AsA content (Fig. 7B). Correlation 74.5%, and 77.4% for HPPD, VTE2, VTE3, VTE1, and VTE4 lines. In
analyses were showed significantly negative correlation between dual-gene transgenic lines VTE2 + VTE4 and VTE3 + VTE4, the ratio
target gene expression level and AsA content (r = À0.91, was 78.7% and 78.4%, respectively (Fig. 6C).
P = 0.00065, n = 50).
3.6. Halliwell–Asada cycle is activated in transgenic lines
3.5. Tocopherol accumulation and ascorbate decrease may affect
glutathione pool and its reductive ratio in Arabidopsis leaves Decrease in ascorbate pool might either be influenced by a
decrease in biosynthesis of AsA or an increase in AsA catabolism. In
Because vitamin C was related with glutathione in plant order to test this hypothesis, the expression levels of enzymes in
physiology, the content of glutathione and reduced glutathione AsA metabolism were studied. In plant cells, AsA related metabolic
composition were observed in the same line. It was observed that network can be divided into two categories: biosynthetic pathway
the change of glutathione content had similar trends with vitamin and Halliwell–Asada cycle. AsA biosynthetic pathway includes

7. 318 Y. Li et al. / Plant Science 178 (2010) 312–320
Fig. 8. Gene expression level of enzymes of AsA metabolic pathways and Halliwell–Asada cycle in single transgenic lines. Y axis, relative mRNA expression level. (A–C) DHAR,
MDAR, and APX expression level in transgenic lines. (D) Tocopherol for reactive oxygen species scavenging and its recycle pathway with Halliwell–Asada cycle. APX, DHAR,
and MDAR are important enzymes of Halliwell–Asada cycle. AsA can reduce tocopherol radical to tocopherol with the help of APX, and itself is oxidized to MDA. With the help
of DHAR and MDAR, MDA can be recycled to AsA. AsA is the protector of tocopherol in plant cells. Data represent the means Æ SD of 50 individual plants.
enzymes GDPME, GDPMPPase, VTC2, L-GalPPase, L-GalDH, and L- leaves of tobacco [30]. In this study, homologous over-expression
GLDH. Halliwell–Asada cycle includes enzyme APX, DHAR, and of HPPD could increase 43% total vitamin E content in leaves. This
MDAR. In order to investigate which step or steps of AsA result was consistent with Tsegaye et al. [31], who over-expressed
metabolism is regulated, the expression of key enzymes of the homozygous HPPD in Arabidopsis resulted in up to a 37% increase in
pathways mentioned above was therefore measured by real-time leaf tocopherol levels. There might be two reasons for slight
PCR. increase in vitamin E content. Firstly, the effect of HPPD gene over-
Expression levels of AsA biosynthetic pathway related genes did expression may be counteracted by the limited expression level of
not show significant differences between NC and transgenic lines. downstream genes in tocopherol biosynthetic pathway, and the
It was suggested that the decrease of ascorbate pool could not be transgenic plant may not have enough capacity to phytylate all the
attributed to the down-regulation of AsA biosynthetic pathway. In increased HGA. Secondly, the increased HGA may be used as
contrast, the expression of genes encoding enzyme APX in substrate for other secondary metabolites in plants.
Halliwell–Asada cycle showed significant increases in the VTE2, The condensation of PDP and HGA is catalyzed by the enzyme
VTE1, and VTE4 over-expression transgenic lines (P < 0.01), and the VTE2. Because HGA and PDP are also used in the synthesis of other
expression level of enzymes DHAR and MDAR was also observed secondary metabolites in plants, such as plastoquinones, phyllo-
increasing, but the up-regulated extent was less than APX quinones, and chlorophylls, VTE2 has important effects on
expression. The VTE3 over-expressing lines presented significant tocopherol pathway at the branch-point from these plastid
increase in the expression of APX compared with NC (P < 0.05), phytyllipid pathways [10,32]. In previous study [33], VTE2 over-
while the expression of DHAR and MDAR showed no significant expression resulted in a 4.4-fold increase in total tocopherol
change. In HPPD over-expression lines, there was no difference in content relative to wild type in the best transgenic line. In this
expression level of the Halliwell–Asada cycle enzymes (P > 0.05) study, over-expression of VTE2 would increase total vitamin E to
(Fig. 8A–C). (29.71 Æ 4.31) pmol mgÀ1 (FW) in average. Among fifty independent
transgenic lines, the best line produced 44.71 pmol mgÀ1 (FW)
4. Discussion vitamin E in leaves. This result seemed lower compared with
Collakova and DellaPenna’s report [33], because all of our results were
4.1. Evaluating functions of tocopherol biosynthetic pathway compared with non-transgenic control, which were plants trans-
enzymes in the view of metabolic engineering formed with the empty vector used in this study. In this way, the
different tocopherol biosynthetic pathway genes could be compared
In order to value the contribution of each enzyme in tocopherol under the same genetic background. a-Tocopherol composition in the
biosynthetic pathway, genes were constitutively over-expressed in best transgenic line was about 85.7%, showed slight decrease
Arabidopsis. Elucidating changes of vitamin E contents or a- compared with NC. In previous report [33], the transgenic lines with
tocopherol composition in transgenic lines were discussed below, highest total tocopherol content had an increase in g-tocopherol
based on the detailed dissection of the biosynthetic pathway. composition. Both results showed the downstream enzyme VTE4 had
HGA is produced from p-hydroxyphenylpyruvate (HPP) by the limitation, which suggested co-expression of VTE4 with VTE2 might
cytosolic enzyme HPPD. It was reported that heterologous over- more obvious effect on a-tocopherol composition.
expression of the barley HPPD gene under control of the 35S The enzymes VTE3, VTE1, and VTE4 have functions on the
promoter only elevated tocopherol content in seeds but not in proportion of tocopherol forms. The methylation reaction cata-

8. Y. Li et al. / Plant Science 178 (2010) 312–320 319
lyzed by VTE3 determines the number and position of methyl Over-expressing two or more genes of one or several metabolic
groups in the final products – without the catalysis of VTE3, the pathways will have multiple and accumulative functions in
tocopherol forms will be d- or b-; otherwise, it will be g- or a- increasing flux of target product, which might be more efficient
tocopherol. Sole over-expression of VTE3 would not remarkably than transgenic plants harboring only one transgene. This result
affect total vitamin E contents in this study, because the a- and g- provides a feasible strategy for metabolic engineering to cultivate
tocopherol composition in Arabidopsis leaves is much higher than plants with higher nutrition values.
b- and d-tocopherol composition. VTE4 plays important role on
producing of a-tocopherol. Over-expression of VTE4 gene solely 4.2. Vitamin E accumulation affected vitamin C and glutathione pools
could elevate a-tocopherol composition in Arabidopsis leaves, and in plant cells
result in 20–30% increase in total vitamin E content. In previous
study [13], over-expression of VTE4 gene in Arabidopsis seeds The opening question in metabolic engineering is whether the
altered the tocopherol composition but not the total tocopherol accumulation of target product might have effects on plant
content. However, in this study total vitamin E content showed physiology or other metabolic products. AsA is important for plant
modest increase. Transcription data showed that genes encoding physiology and nutrition. Glutathione is water-soluble antioxidant
upstream enzymes of tocopherol biosynthetic pathway, especially in plant, which was related with AsA by Halliwell–Asada cycle. In
VTE2, had slightly elevated expression level in VTE4 transgenic line. this study, content and reductive ratio of AsA and GSH was
It seems that the pulling force of the last enzyme in biosynthetic analyzed in transgenic plants, respectively. Our study proved that
pathway helped to induce the potential ability of the upstream in single-gene transgenic lines, the accumulation of vitamin E
enzymes. However, the endogenous expression of VTE2 was content or change in a-tocopherol composition, resulted in a
limited [33], which gave hints that the combination of VTE2 and decrease in total ascorbate pool and a modest increase in the
VTE4 might have both vitamin E content and a-tocopherol reductive ratio of AsA. Glutathione had similar changing trends
composition elevated. The significant change in VTE1 over- with AsA. In multiple-gene transgenic lines, whose vitamin E
expression lines was the increase of g-tocopherol and its isoforms content and composition was both changed as described detailed
(such as plastochromanol-8, the natural homologue of g-toco- above, the decreasing in AsA and GSH was also observed which
trienol), which increased by 5.5-fold in average compared with NC, seemed to have lower contents than single-gene transgenic lines.
while a-tocopherol did not show significant increase. The All those observation indicated that vitamin E accumulation in
accumulation of g-tocopherol and its isoforms might be resulted plant cells might have effect on vitamin C and glutathione. It
from the limitation of endogenous VTE4, and this result was seemed that the lines with higher vitamin E content would have
similar with report from Kanwischer et al. [22], who increased total lower contents AsA and GSH. In summary, increasing in vitamin E
tocopherol levels of at least 7-fold in Arabidopsis leaves. The levels triggered compensatory changes in ascorbate and glutathi-
different increase extents might be the different plant expression one levels, which was also demonstrated in former studies [22].
vectors and promoters used in different studies. Because tocopherols, ascorbate and glutathione could be
To further study the effect of co-operation of key enzymes on related with Halliwell–Asada cycle, further study suggested the
vitamin E content and composition, dual-gene co-expression was changing of antioxidants might be affected by the activity of
conducted. Based on the results of single-gene over-expression Halliwell–Asada cycle in transgenic lines.
lines, two different combinations (VTE2 + VTE4 and VTE3 + VTE4) Tocopherol and Halliwell–Asada cycle are closely associated in
were studied. In VTE3 + VTE4 lines, although vitamin E content chloroplasts. Tocopherol can protect PUFA and hydrosulfide
could not be obviously elevated, nearly all other tocopherol groups of proteins from oxidization with itself oxidized to
forms switched to a-tocopherol. It was found that the tocopherol radical [34]. Plants can convert the tocopherol radicals
VTE2 + VTE4 lines could increase vitamin E content and enhance back in time with the help of Halliwell–Asada cycle (Fig. 8D). The
a-tocopherol proportion at the same time. Vitamin E content in process can be generalized as follows. Tocopherol radical is
VTE2 + VTE4 lines was higher than in VTE2 or VTE4 single reduced to tocopherol by AsA with the help of APX, and AsA is
transgenic lines. It seems that the pulling force of the oxidized into monodehydroascorbic acid (MDA). MDAR reduces
downstream enzyme (the faucet enzyme) VTE4 plays a more MDA into AsA. On the other hand, dehydroascorbic acid (DHA) is
important role in stimulating vitamin E accumulation whereas spontaneously produced from MDA and can be reduced to AsA by
the functioning of the upstream enzyme VTE2 is increased DHAR, with GSH oxidized to GSSG [35].
proportionally. Although VTE2 is the enzyme which catalyzes the AsA plays important fuction in Halliwell–Asada cycle and is
condensation of HGA and PDP, its function seems flux-limiting directly related with tocopherol. GSH participates Halliwell–Asada
and hence represents the first committed step in tocopherol cycle and helps AsA regenerate. From the view of the overall
biosynthesis. VTE4 catalyzes the final step of a-tocopherol, network, redox homeostasis is existed in plants which can keep the
which is not inhibited by end product, it is suggested that it is balance between reactive oxygen species (ROS) and antioxidants
completely desirable to fundamentally enhance the a-tocopher- [36]. In this study, genetic engineering increased target gene
ol production. In previous report [33], seeds of VTE2 + VTE4 dual expression level, activated tocopherol biosynthesis, and the
over-expression lines had a 12-fold increase in vitamin E activity increased tocopherols might active Halliwell–Asada cycle and
relative to wild type [Vitamin E activity was calculated as one affect the antioxidants associated with it, such as AsA and GSH.
milligram of a-, b-, g-, and d-tocopherol corresponds to 1, 0.5, However, it was reported that the amount of endogenous DHAR
0.1, and 0.03 mg of a-TE, respectively (Food and Nutrition Board, was limited [36], and APX showed higher expression level than
Institute of Medicine, 2000)]. In other study we did in lettuce, the MDAR and DHAR in transgenic lines in this study, which could
VTE2 + VTE4 combination had a 19-fold increase in vitamin E accelerate the oxidization process of AsA to MDA and DHA. The
activity relative to non-transgenic control (unpublished). All oxidized form of AsA would be quickly degenerated, resulting in
these data indicated that VTE2 + VTE4 dual transgenic lines are the total ascorbate pool decreasing. On the other side, the genes of
crucial and useful for metabolic engineering. Also based on the AsA biosynthetic pathway were not up-regulated in transgenic
single-expression and dual expression data, triple-expression of plants. As a result, the level of endogenous AsA might be
VTE2, VTE1 and VTE4 is considered in leaves of lettuce and seeds decreasing. It indicated that elevating the activity of AsA
of Brassica campestris L. in our lab, and it is looking forward to biosynthetic pathway might complement the decreasing in AsA
more expectant results. content.

9. 320 Y. Li et al. / Plant Science 178 (2010) 312–320
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