ABBS 2009,41(02):

Pdf file on Regulation of polyphenols accumulation by combined
overexpression/silencing key enzymes of phyenylpropanoid pathway

Junli Chang^#, Jie Luo^#, and
Guangyuan He*

China-UK HUST-RRes Genetic Engineering and
Genomics Joint Laboratory, Key Laboratory of Molecular Biophysics, Ministry of
Education, College of Life Science and Technology, Huazhong University of
Science and Technology (HUST), Wuhan 430074, China

#These authors contributed equally to this work

*Corresponding author: Tel, 86-27-87792271; Fax,
86-27-87792272; E-mail, [email protected]; [email protected]

Abstract:

There is a growing interest in the metabolic
engineering of plant with increased desirable polyphenols such as chlorogenic
acid (CGA) and rutin. In this study, the effects of overexpression of both
phenylalanine ammonia lyase (AtPAL2), the first enzyme of the phenylpropanoid
pathway and hydroxycinnamoyl-CoA quinate:hydroxycinnamoyl transferase (NtHQT),
the last enzyme of CGA biosynthesis, and the overexpression of AtPAL2 together
with silencing of NtHQT were investigated in tobacco. Transgenic tobacco plants
overexpressing AtPAL2 showed 2 and 5 times increases of CGA and rutin levels
than the wild type plants respectively. Overexpression of NtHQT further
increases the accumulation of CGA in the AtPAL2 plants to about 3 times than
that of the wild type level, while silencing of NtHQT in AtPAL2 plants results
in about 12 times increase of rutin level than that of the wild type plants.
Simultaneous overexpression of phenylalanine ammonia lyase (PAL) and
overexpression/silencing HQT could be used for the production of functional
food with increased polyphenols.

Keywords 
phenylalanine ammonia lyase; hydroxycinnamoyl-CoA quinate:
hydroxycinnamoyl transferase; phenylpropanoid; metabolic engineering

Received: September 8, 2008        Accepted: November 19, 2008

Introduction

The phenylpropanoid pathway (Fig. 1) is
responsible for the synthesis of a large range of low molecular weight
polyphenolics which occur naturally in plant tissues, including flavonols,
flavones, flavanones, flavanones, catechins, anthocyanins, isoflavonoids,
dihydroflavonols, and stilbenes [1]. To date, more than 4000 flavonoids
have been identified. As a group, flavonoids are involved in many aspects of
plant growth and development, such as pathogen resistance, pigment production,
UV light protection, pollen growth, and seed coat development [2]. There is increasing evidence to
suggest that flavonoids are health-protecting components in the human diet as a
result of their high antioxidant capacity [3-6]. This is also supported by their
ability to induce human protective enzyme systems [7-9], and their protection against major
diseases such as cardiovascular diseases, cancer [10] and age-related diseases such as
dementia [11]. In addition, several epidemiological
studies have suggested a direct relationship between cardioprotection and
consumption of flavonols from dietary sources [12].

Chlorogenic acid
(CGA) is the major soluble polyphenol in a lot of plants such as tobacco,
tomato, and potato. It also accumulates to substantial levels in apples, pears,
and coffee. Inhibition of CGA accumulation in tobacco results in accelerated
cell death in mature leaves, typical of oxidative stress and increased levels
of the oxidized lipid malondialdehyde [13]. Because of its
high bioavailability, CGA is probably more accessible than that of many other
flavonoids as a potential antioxidant from plants. CGA can also limit
low-density lipid (LDL) oxidation, the major determinant of the initial events
in atherosclerosis. Furthermore, it removes particularly toxic reactive
substances by scavenging alkylperoxyl radicals and may prevent carcinogenesis
by reducing the DNA damage they cause [14]. Recent research
also suggested the responsibility of CGA for the reduction of diabetes [15]. CGA is
therefore an important and yet somewhat overlooked dietary bioactive.

Another major and
important polyphenol in plants is rutin (quercetin-3-O-rutinoside), which
widely exists in many crops and fruits. Lopez et al.
reported that quercetin had the ability to reduce blood pressure and
endothelial dysfunction in animal models of hypertension [16]. The hypertension and oxidative stress
prevention, and the vascular protection effects by chronic treatment of
quercetin were also reported [17].

Based on these types of studies, there is growing
interest in strategies to increase the polyphenol levels such as CGA and rutin
have may have potential to develop functional foods which enriched with
health-protective polyphenols.

Phenylalanine
ammonia lyase (PAL; E.C. In this paper, the effects of overexpression of
both AtPAL2 and NtHQT, and overexpression of AtPAL2 and silencing of NtHQT
on the accumulation of different phenylpropanoids in tobacco were investigated.
Transgenic plants with increased PAL activities had higher
accumulations of both CGA
and rutin, which led to higher antioxidant capacity and enhanced tolerance to
oxidative damage. The accumulation of CGA was even higher when NtHQT was
overexpressed in the presence of AtPAL2,
while decreased accumulation of CGA together with increased accumulation of
rutin was achieved when NtHQT was
silenced in the presence of AtPAL2
than in the plants that only overexpressing AtPAL2.
This strategy can be used for the development of functional foods that offer
protection against cardiovascular disease and cancer through diet.

Materials and
Methods

Materials

Nicotiana tobacum
Samsun NN, Arabidopsis thaliana (Columbia ecotype), and Agrobacterium
tumefaciens LBA4404 were used in this study.

AtPAL2
overexpression vector construction and transformation

Total
RNA was extracted using the TRI-REAGENT (Sigma-Aldrich, St. Louis, USA) from
flowers of Arabidopsis thaliana (Columbia ecotype) plants according to the
procedures provided by the manufacturer. First strand cDNAs were synthesized by
reverse transcription kit (TaKaRa, Dalian, China), and open reading frame (ORF)
of AtPAL2 was amplified using primers P1: 5‘-attB1-CCACCATGGATCAAATCGAAGCAATGTTG-3‘ and P2: 5‘-attB2-TAGCAAATCGGAATCGGAGCTG-3‘ and cloned into the Gateway Donor
vector (pDONR207) (Invitrogen, Carisbad, USA) by BP reaction. The resulting
vector pDONR207-AtPAL2 was then used to do the LR reaction to put AtPAL2 into Gateway compatible
destination vector to create the overexpression vector pJAM1502-AtPAL2.
Similarly, the ORF of NtHQT was
amplified from tobacco cDNA using primers P3: 5‘-attB1-CCACCatgggaagtgaaaaaatgatga3‘ and P4: 5‘-attB2-tcaaaattcatacaaatacttc-3‘ and eventually into pJAM1502-NtHQT.
The gene silencing plasmid pFRN-NtHQT for NtHQT
silencing was described previously [19]. The resulting plasmids were verified and used for stable
transformation of tobacco using A. tumefaciens strain LBA4404 by the
method described previously [20]. The transformed plants were selected on kanamycin.

Northern
blot analysis

Total
RNA was extracted and purified from tobacco leaves according to the procedures
provided by manufacturer as mentioned above (Sigma-Aldrich). The resulted RNA
was then separated on denaturing agarose gels and transferred onto nylon
membranes (GE Healthcare, Piscataway, USA) and hybridized to radioactive DNA
probes as previously described [19].

Determination
of enzyme activities

PAL
activity was assayed using the method as previously described [21] with slight modification. Briefly, the assay was carried out in a
reaction mixture containing

Measurement
of Anthocyanin

Anthocyanin
was extracted from petals of control and transgenic tobacco plants and measured
by the method of Martin et al. [23]

To
generate AtPAL2/NtHQT lines, AtPAL2 overexpression line P3 was
crossed to NtHQT line HO3.
F1 seeds of AtPAL2/NtHQT plants were
sowed on Murashige and Skoog (MS) plates containing 100 mg/l kanamycin
and screened for the incorporation of both 35S-AtPAL2 and 35S-NtHQT by PCR
amplification of genomic DNAs. Seedlings showed positive results in both PCR
amplifications (AtPAL2/NtHQT lines)
were transferred into soil and used for polyphenol analyses. Similarly, AtPAL2/Nthqt
lines were generated between AtPAL2
overexpression line P3 and NtHQT
silencing line HS2, screened for 35S-AtPAL2 and CHSA-NtHQT insertion and then
analyzed for polyphenols. AtPAL2
overexpression line P3 was backcrossed with the wild type (WT) tobacco (Samsun
NN), F1 seedlings that were kanamycin resistant and positive for AtPAL2 insertion (AtPAL2 lines) were transferred into soil and used as control
plants. For polyphenol analyses, leaf samples from 12 plants of each line were
pooled and analyzed for their CGA, rutin contents by HPLC.

Results

Generation
of transgenic plants

The
vectors used for the generation of the transgenic lines in this study were
shown in Fig. 2(A). Tobacco (N. tobacum Samsun NN) was transformed
with a binary vector (pJAM1502-AtPAL2) containing the Arabidopsis PAL2 gene under the control of the
constitutive cauliflower mosaic virus (CaMV) double 35S promoter (see Materials
and methods) vis Argobacterium mediated transformation to get AtPAL2 overexpression lines [Fig. 2(B,C)]. Similarly, pJAM1502-NtHQT
and pFRN-Nthqt were introduced into tobacco to obtain NtHQT overexpression [Fig.
2(D,E)] and Nthqt silencing
[Fig. 2(F,G)] lines, respectively.
More than thirty independent transformants for each constructs were produced.
After confirming the insertion by PCR, the transcription of the All these
plants were further detected for their transcription levels of AtPAL2 for AtPAL2 overexpression NtHQT
for NtHQT overexpression/silencing
plants, respectively by northern
blot [Fig. 2(B,D,F)]. Fig. 2(B) showed that the WT tobacco
had no hybridizing band under washing conditions used in this study. This
suggests that sequence similarity of the endogenous tobacco gene to the
Arabidopsis gene is relatively low and that any bands detected in the AtPAL2 transgenic plants are due to the
presence of the Arabidopsis gene only. The target fragments were obtained from
most of the transgenic plants detected by PCR amplification but not the control
plant. We could not detect any AtPAL2
expression in line P2 and P4. This might because the level of AtPAL2 expression in this line was too
low to be detected by northern blot, or because of the silencing of the foreign
gene after integration. To confirm the equal loading of RNA samples, RNA gel
blot were also hybridized with a probe encoding the ubiquitin from tobacco. As
shown in Fig. 2(D), line HO3, HO4
and HO2 of the NtHQT overexpression plants had increased NtHQT transcription levels than that of the wild type plants,
indicating the overexpression of NtHQT
in these lines. In contrast, decreased NtHQT
transcriptions were detected among most of the NtHQT silencing lines tested with HS2 and HS3 showed the lowest
transcription levels of NtHQT [Fig. 2(F)]. To further confirm the
above transgenic lines at the protein level, three independent lines that
showed the highest overexpression/silencing effects were used for further
enzyme activity assays. The leaves of the AtPAL2
overexpression (line P1, P3, and P5), NtHQT
overexpression (line HO2, HO3, and HO4) and NtHQT
silencing (line HS1, HS2, and HS3) plants were determined for their PAL or HQT
enzyme activities, and the results were given in Fig. 2(C,E,G), respectively. It could be seen that the PAL
activities were correlated with the expressions of AtPAL2 with the highest PAL activity obtained from leaves of P3,
which was more than 3.5 times over the control levels. The highest NtHQT overexpression lines HO3, HO4 had
about 2.8 and 2 times activities than that of the control while the remaining
HQT activities in the NtHQT silencing
lines HS1 to HS3 were about 10% (7% to 10%) of the control plants,
respectively.

Overexpression
of AtPAL2 led to the increase of both CGA and rutin in tobacco leaves

The
transgenic tobacco plants overexpressing AtPAL2 had normal visual
phenotype, growth characteristics, and fertility compared the wild type plants.
However, when leaves of control and transgenic plants were extracted (with The
effect of AtPAL2 overexpression on
antioxidant capacity was determined by TEAC assay, and the results were shown
in Fig. 3(D). Hydrophilic TEAC
increased with the increased AtPAL2
overexpression. The highest hydrophilic TEAC of 23.6 mmol Trolox per gram dry
weight (DW) was obtained in AtPAL2
overexpression line P3, which was about 2.5 times that of the control level. In
contrast to the hydrophilic TEAC, no increase of hydrophobic TEAC was seen
between the control and the transgenic plants, suggesting that the major
effects of AtPAL2 overexpression were
restricted to the increase of hydrophilic substrates such as simple phenolics
or flavonoids, with no effect on hydrophobic compounds (e.g. carotenoids,
tocopherol). Measurement
of anthocyanins extracted from tobacco flowers showed that control and
transgenic plants had similar anthocyanin contents in their flowers [Fig. 3(E)],
suggesting that metabolic flux in the anthocyanin biosynthesis pathway was not
affected by the introduction of AtPAL2.

The
above results showed that overexpression of AtPAL2
could direct the flux into different branches of the phenylpropanoid pathway
and resulted in increased accumulation of both CGA and rutin in tobacco leaves.

Overexpression
of NtHQT further increased CGA production in AtPAL2 plants

To
investigate whether the production of CGA can be further increased, tobacco
plants harboring AtPAL2 were crossed
with plants overexpressing NtHQT (line
P3 crossed with line H3, see Materials
and Methods). Fig. 4 showed
that CGA accumulation in the AtPAL2/NtHQT
overexpressing lines reached about 46.2±5.3 mg/g DW, which was about 1.4 times
higher than the AtPAL2, and 3.0 times
the wild type plants (data not shown). The average rutin content in the AtPAL2/NtHQT plants reached a level of
3.5±0.5 mg/g DW, which was of no difference to the amount of rutin accumulated
in the AtPAL2 overexpression lines.

Gene
silencing of NtHQT in AtPAL2 helped to increase rutin production

To
determine the effects of silencing of NtHQT
in the AtPAL2 overexpression plants, NtHQT silencing plant (line HS2) was
crossed with AtPAL2 (line P3) (see Materials and Methods), and the CGA
and rutin contents in the leaves were shown in Fig. 5. The CGA content (14.8±2.0 mg/g DW) in the AtPAL2/Nthqt
line was about 51 percent that of the AtP4AL2/NtHQT lines, while the rutin content
reached 8.4±1.1 mg/g DW, which was 2.8 times higher than that in the AtPAL2 lines.

Discussion

Products of the phenylpropanoid pathway are
structurally and functionally diverse and are synthesized in response to both
developmental and environmental cues [25–27]. PAL catalyze the first step of the
phenylpropanoid pathway, and is typically encoded by a small multigene family.
In species such as Oenothera, only one of the two isoform of PAL was involved
in flavonoid biosynthesis [28], which suggested that in species
possessing multiple PAL isoforms, the flux into various branches of
phenylpropanoid pathway might be regulated by these isoforms either
individually or coordinately. In addition, the fact that individual members of
the PAL gene family are expressed differently during plant development
and in response to different stress stimulus suggested that certain PAL genes
may associate preferentially with specific multienzyme complexes to control the
flux of metabolites through the different branches of the phenylpropanoid
pathway [29]. Despite of all this investigations
and suggestions, the metabolic significance of these is usually unknown. In Howles’
report [18], overexpression
of bean PAL2 gene resulted in increased level of CGA but not rutin. We
reported here the
overexpression of AtPAL2 gene resulted in the increase of both CGA and
rutin in transgenic tobacco. The results that we got here gave the first time
direct evidence of the idea that although functional redundancy might exist for
the PAL family, different PAL gene (or different isoforms of PAL
gene) might be involved into different branches of phenylpropanoid pathway.

Increasing
evidence of healthy related functions of polyphenols in dietary has made
metabolic engineering of phenylpropanoid pathway the subject of investigation
in recent years [27]. A number of enzymes and the genes encoding these enzymes had been
cloned and their roles had been investigated. So far most of the researches
concerned the alternation of flux distribution among different branches this
pathway. Since the total flux into this pathway is unchanged [30], the increase of flux into one branch of the pathway will normally lead
to the decrease of flux into other branches within this pathway [31]. The advantage of overexpression of PAL is that it can
increase the total flux into this pathway, so simultaneously increased
accumulation of bioactives in different branches of this pathway could be
achieved (Fig. 3). We also
demonstrated that by combined overexpression of PAL with the
overexpression/silencing of a specific branch of the pathway, further increase
of the specific bioactive(s) can be obtained. These strategies can be used as
for the metabolic engineering of multi-branch pathway(s) such as the
phenylpropanoid pathway to produce functional food with increased polyphenols.

Funding

This work was
supported by a grant from the National Science Foundation of China (No.
30500038 to J.L.).

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