Transgenic Tobacco Overexpressing Tea cDNA Encoding
Dihydroflavonol 4-Reductase and Anthocyanidin
Reductase Induces Early Flowering and Provides Biotic
Stress Tolerance
Vinay Kumar1, Gireesh Nadda2, Sanjay Kumar1, Sudesh Kumar Yadav1*
1 Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, Himachal Pradesh, India, 2 HATS
Division, CSIR-Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur, Himachal Pradesh, India
Abstract
Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and
anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have
generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to
evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of
CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content
of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in
transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress
was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco
overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura.
Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and
antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and
biotic stress tolerance.
Citation: Kumar V, Nadda G, Kumar S, Yadav SK (2013) Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin
Reductase Induces Early Flowering and Provides Biotic Stress Tolerance. PLoS ONE 8(6): e65535. doi:10.1371/journal.pone.0065535
Editor: Alfredo Herrera-Estrella, Cinvestav, Mexico
Received December 13, 2012; Accepted April 22, 2013; Published June 18, 2013
Copyright: ß 2013 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Council of Scientific and Industrial Research (CSIR), GOI under NMITLI program (TLP003). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: sudeshkumar@ihbt.res.in
shown in Figure 1. Flavan-3-ols biosynthesis shares anthocyanidin
biosynthesis pathway from phenylalanine to leucoanthocyanidin
(flavan-3, 4-diol). In this pathway, DFR (dihydroflavonol 4reductase; EC 1.1.1.219) catalyzes the production of leucoanthocyanidin. Leucoanthocyanidin is a common substrate for the
production of both flavan-3-ols and anthocyanins, and is
converted to anthocyanidin by anthocyanidin synthase (ANS;
EC 1.14.11.19) and to flavan-3-ols (catechin) by leucoanthocyanidin reductase (LAR; EC 1.17.1.3). Anthocyanidin is converted to
epicatechin by anthocyanidin reductase (ANR; EC 1.3.1.77) [2,3].
The DFR and ANR have been considered aspivotal enzymes of
flavan-3-ols biosynthesis. They belong to the short chain
dehydrogenase/reductase or DFR super family. By sequence
similarity, both are closely related to each other [7]. Genes
encoding these two enzymes are characterized by a similar exon/
intron pattern and enzymatic proteins contained an amino acid
sequence motif for NADPH binding. The DFR has been identified
and characterized from several plant species [8–11]. The
transgenic tobacco overexpressing DFR encoding cDNA from
Medicago truncatula (MtDFR) has been produced and studied with
respect to flavonoids content [12]. Also, the transgenic rice
overexpressing MtDFR has been reported for altered metabolites
profile [13]. Similarly, ANR has also been identified and
Introduction
Flavonoids comprise one of the largest groups of plant
secondary metabolite. These are widespread throughout the plant
kingdom and are found to be accumulated in different organs and
tissues of plants. They are mainly involved in providing protection
to plants against predation, pathogen (bacteria and fungi) attack,
and act as effective repellant and prevent feeding by herbivores
[1]. They are the major quality factors for forage crops. The
higher concentration of flavonoids can decrease the palatability of
forage crops. In some forage crops, the presence of flavonoids can
also be a positive trait and recognized as health beneficial
compounds to the ruminant animals by reducing pasture bloat [2–
5]. Several different classes of flavonoids including anthocyanins,
flavonols, isoflavones, monomeric flavan-3-ols (catechins and
epicatechins) and oligomeric flavan-3-ols (proanthocyanidins;
PAs) contribute to the growth and survival of plants under UV
exposure as well as against pathogen and herbivores [4]. At the
same time, they impart astringency and flavor to beverages such as
wine, fruit juice and tea [6].
The genetics and biochemistry of flavonoid biosynthetic
pathway has been extensively studied in number of plant species.
The broad outline of flavonoid biosynthesis pathway in plants is
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CsANR and CsDFR Transgenic Tobacco
NADPH-dihydromycertin reductase; EC 1.1.1.219) has been
cloned from tea cultivar UPASI 10. CsDFR is 1,413 bp full-length
cDNA with ORF of 1,044 bp (115–1158) and encoding a protein
of 347 amino acid residues [10]. Also, a cDNA encoding for
anthocyanidin reductase (ANR: Flavan-3-ol: NAD(P)+ oxidoreductase: EC 1.3.1.77) has been cloned from tea cultivar UPASI 10.
CsANR is 1,233 bp full-length cDNA with ORF of 1,014 bp (79–
1092) and encoding a protein of 337 amino acid residues. The
CsANR cDNA from tea cultivar UPASI 10 showed 97% and 80%
homology at nucleotide level with previously reported CsANR2
(Accession
no. GU992400)
and
CsANR1
(Accession
no. GU992402) from blister blight-resistant tea cultivar
TRI2043, respectively. Hence, this has suggested that CsANR
used in this study is CsANR2. Interestingly, CsANR activity has
characterized from several plant species [14–18]. Transgenic
tobacco overexpressing MtANR has been generated and analyzed
for its effect on PAs content [19]. The PAs accumulation was also
reported to be altered by suppressions of ANR1 and ANR2 in
Glycine max [20]. Recently, the proteins encoded by ANR1 and
ANR2 genes from leaf tissue of a blister blight-resistant tea cultivar
TRI2043 have been documented for different level of epimerase
activity and exhibited similar kinetic properties [21].
Flavan-3-ols are the building block of PAs. They are also
reported for their antioxidant potential and free radical scavenging
activity [5,6]. Tea (Camellia sinensis) plant contains an extraordinarily high ,25–30% of flavan-3-ols on leaf dry weight basis.
Recently, progress has been made towards elucidation of flavonoid
biosynthesis in tea. A cDNA encoding for DFR (Flavan-3-ol
Figure 1. General outline of anthocyanins and flavan-3-ols biosynthetic pathway in plants. The enzymes are: PAL, Phenylalanine
ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H flavanone-3hydroxylase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanin synthase; ANR1, anthocyanin reductase1; ANR2,
anthocyanin reductase2; GT, Glucosyl transferase.
doi:10.1371/journal.pone.0065535.g001
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CsANR and CsDFR Transgenic Tobacco
been found to be directly correlated with epicatechin content in
tea [17].
Previously, a number of transgenic plants have been developed
and characterized with respect to engineer flavonoid biosynthetic
pathway [15,20,22–26]. Since tea is reported to contain very high
content of flavonoids particularly flavan-3-ols, the genes encoding
enzymes of regulatory steps of flavonoid pathway from tea were
used in this study to engineer tobacco. Recent studies have
described that CsF3H, CsANR and CsDFR encoding enzymes are
crucial for flavonoid biosynthesis in tea [10,17,27]. Among them,
CsANR and CsDFR belong to DFR super family. In view of all
this, present study was planned to generate transgenic tobacco
overexpressing tea cDNA CsDFR and CsANR encoding for DFR
and ANR. To see the influence of CsDFR and CsANR cDNA
overexpression, transgenic tobaccos were analyzed for flavonoids
content and antioxidant potential. They were further assessed for
the influence of these transgenes on their development and
protective abilities against biotic stress.
oligo dT12–18, 200 U of superscript III RT, and 10 mM dNTPs in
a 20 ml reaction volume. Equal quantity of cDNA was used as
template in PCR with gene-specific primer sets for probing the
expression of NtPAL, NtC4H, Nt4CL, NtCHS, NtCHI, NtF3H,
NtFLS, NtDFR, NtANR1, NtANR2 and NtANS genes. All primer
sequences used in this study are shown (Table S1). The primers of
NtANR1 and NtANR2 were designed in such a way that RT-PCRs
could be performed specifically. Linearity between the amount of
input RNA and the final PCR products was verified and
confirmed. After standardizing the optimal amplification at
exponential phase, PCR was carried out with the conditions of
94uC for 4 min for 1 cycle, 94uC for 30 s, 52uC (NtANS), 54uC
(NtC4H, Nt4CL, NtCHS, NtANR1, NtANR2), 58uC (NtDFR, NtPAL,
NtCHI, NtF3H and NtFLS) for 30 s, and 72uC for 30 s for 27
cycles. The amplified products were separated on 1% agarose gel
and visualized with ethidium bromide staining. A gel documentation system (Alpha DigiDocTM, Alpha Innotech, USA) was used
to scan the gel and changes in the gene expression were analyzed
by calculating integrated density values (IDV) using AD-1000
software. The 26S rRNA-based gene primers were used as internal
control for relative gene expression studies [29].
Materials and Methods
Preparation of pCAMBIA-CsDFR and pCAMBIA-CsANR
construct, tobacco transformation and transgenic
confirmation
Measurement of morphological and yield parameters
The parameters namely days to flowering, capsules number,
seed yield and thousand seed weight were analyzed. These
parameters were analyzed in eight plants of each selected lines of
CsDFR and CsANR overexpressing tobaccos as well as control
tobacco plants. The capsules were counted at their maturation
time and seed yield and weight of thousand seeds was measured
after harvesting the capsules of CsDFR and CsANR overexpressing
transgenic lines as well as control tobacco plants.
The cDNA sequence of CsDFR and CsANR is available at NCBI
with Accession number AY64027 and AY641729, respectively.
The isolated cDNAs of CsDFR and CsANR from C. sinensis cultivar
UPASI-10 were cloned into pCAMBIA 1302 plasmid between Nco
I and Bgl II restriction sites. This was resulted in the formation of
recombinant construct pCAMBIA-CsDFR and pCAMBIA-CsANR.
These constructs were transferred into Agrobacterium tumefaciens
strain LBA4404 by triparental mating. A. tumefaciens harboring
pCAMBIA-CsDFR and pCAMBIA-CsANR constructs were used
for leaf disc transformation of tobacco (Nicotiana tabacum cv Xanthi
nc) following the standard transformation protocol [28]. Plants
rooted on hygromycin selection (50 mg/ml) were screened for the
presence of CsDFR and CsANR transgene by carrying out PCR
with genomic DNA and gene specific primers (CsDFR forward
primer 59-ATGGAAGCCCAACCGACAGCTC-39 and reverse
primer 59-TCAATT CTTCAAAATCCCCTTAGCCT -39;
CsANR forward primer 59-ATGAAAGACTCTGTTGCTT
CTGCC-39 and reverse primer 59-TTA AACCTTGTTGCC
ATTGACAGG-39). The reaction conditions were as follow: 94uC
for 1 min, and then 35 cycles of 30 s at 94uC, 30 s at 54uC (for
CsDFR) and 58uC (for CsANR), and 2 min at 72uC. PCR products
were separated by agarose gel electrophoresis and visualized with
ethidium bromide. T1 transformants were self-pollinated and the
seeds obtained from T1 were analyzed for segregation by
germinating on half strength Murashige and Skoog medium
supplemented with hygromycin (50 mg/ml). One month old
plants were transferred in green house in pots containing the
manure, sand and soil in 1:1:2 ratios. Tobacco plant transformed
with pCAMBIA vector having no transgene CsDFR or CsANR was
used as control (mock). The third leaf of three plants of each
independent transgenic line as well as control tobacco plant was
harvested and used for various sample preparation.
Estimation of flavonoids content
The 250 mg of freeze-dried leaf powder of transgenic lines as
well as control tobacco plants was taken for extract preparation.
Total flavonoids content in the extract was determined using the
previously described method [30]. Total flavonoids content was
expressed as mg/g quercetin equivalent. Flavan-3-ols such as
catechin (Cat), epicatechin (EC) and epigallocatechin (EGC) were
analyzed by HPLC method [31]. Briefly, 1 g of tobacco leaf tissue
of each transgenic tobacco line in triplicates was freeze dried, and
used for flavan-3-ols extraction with 70% methanol. Flavan-3-ols
were measured by Merck Hitachi HPLC (Darmstadt Germany)
using C18 Licrocart column (250 mm65 mm65 mm) and absorbance was read at 210 nm. The flavan-3-ols (+)-Cat, (2)- EC and
(2)-EGC were used as standard from Sigma for estimation of
respective constituent.
Estimation of DPPH radical activity
Antioxidant activity of the methanolic extract of leaves of three
plants from each transgenic as well as control tobacco plants was
performed. The ability of plant extract to oxidize stable DPPH
(diphenylpicryl-hydrazyl) free radicals was estimated as described
earlier [32]. The initial absorbance of DPPH in methanol was
measured using spectrophotometer at 517 nm until the absorbance remained constant. A total of 50 ml of extract was added to
1950 ml of 0.1 mM methanolic DPPH solution. The mixture was
incubated at room temperature for 30 min before the change in
absorbance at 517 nm was observed. The percent of inhibition
was calculated using the formula,
RNA isolation and semiquantitative PCR
The 100 mg of leaf tissue of transgenic lines as well as control
tobacco plants was ground in liquid nitrogen and total RNA was
isolated using RNeasy Plant Mini Kit (Qiagen, Germany). The
total RNA from each sample was treated with DNase I to remove
DNA contamination. cDNA was prepared according to manufacturer’s protocol (Invitrogen, USA) using 2 mg of total RNA, 250 ng
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Figure 2. Generation of CsDFR and CsANR transgenic tobacco. A, Graphic representation of pCAMBIA 1302 vector with cDNA of CsDFR and
CsANR. CsDFR and CsANR cDNA was inserted in-between the NcoI and BglII restriction site of pCAMBIA 1302. B, Genomic DNA PCR confirmed the
insertion of CsDFR and CsANR cDNA in plant genome of transgenic lines. C, Semi-quantitative PCR documented the transcript expression levels of
CsDFR and CsANR in transgenic tobacco lines. Housekeeping gene 26S rRNA was used as internal control for expression study and experiments were
repeated at least three times with similar results.
doi:10.1371/journal.pone.0065535.g002
Percent of inhibitionð%Þ~
Percent growth inhibition~
½ðA517 of control{A517 of sampleÞ=A517 of control|100:
ðWeight gain incontrol{
Weight gain in treated=Weight gain in controlÞ|100
Evans blue staining for H2O2-induced cell death
Fresh leaves from three plants of each transgenic line as well as
control tobacco plants were treated with 5 mM H2O2 for 2 h as
described previously [33]. Samples were then incubated with
0.05% Evans blue for 15 min, followed by extensive washing to
remove unbound dye. Dye bound to dead cells was solubilized
with 50% methanol and 1% sodium dodecyl sulfate at 50uC for
30 min and quantified by taking absorbance at 600 nm.
Phytohormone analysis
Endogenous gibberellin (GA3) and auxin in the form of free
indole-3-acetic acid (IAA) content of control as well as transgenic
tobacco lines were determined by HPLC method as described
earlier [35]. Five gram fresh tissue of control as well as transgenic
tobacco lines were homogenized with 70% (v/v) methanol and
stirred overnight at 4uC. The extracts were used for further
extraction with ethyl acetate and diethyl ether stepwise. After that,
samples dissolved in methanol were used for HPLC analysis and
absorbance was read at 210 nm for GA3 and 265 nm for IAA.
The GA3 and IAA from Sigma were used as standard for
estimation of respective constituent.
Leaf disc non-choice test
To check the possible anti-feeding behavior of tobacco
cutworms (Spodoptera litura), leaf discs of 6 cm diameter were
prepared from transgenic lines as well as control tobacco plants
and experiment was performed with some modification as
described earlier [34]. These discs were kept in different petri
dishes containing wet filter paper with 10 larvae (less than 24 h
old) of S. litura and allowed to feed for 7 consecutive days.
Experiment was performed in three replicates for each transgenic
line. The petri dishes were kept at 2762uC, 6065% RH and a
photoperiod of 16 h: 8 h (Light: Dark). The growth behavior of
cutworm caterpillar was represented as percentage growth
inhibition and calculated as
Protein estimation
Total protein content in various extracts was estimated
following the Bradford method [36]. Standard was prepared with
BSA and used for protein estimation.
Results
Confirmation of transgenic tobacco overexpressing
CsDFR and CsANR cDNA
Transgenic tobaccos were generated using Agrobacteriummediated transformation of recombinant construct pCAMBIACsDFR and pCAMBIA-CsANR (Fig. 2A). The integration of
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transgenic lines (Fig. 5A). To check the influence of CsDFR and
CsANR overexpression on flavan-3-ols in tobacco, Cat, EC and
EGC content was estimated by HPLC. Only (+)-Cat, (2)- EC and
(2)-EGC flavan-3-ols were detected in tobacco. The Cat, EC and
EGC were found to be increased in both CsDFR and CsANR
overexpressing transgenic lines. The Cat level was estimated in
range of 0.38–0.5 mg g21 DW in CsDFR and CsANR overexpressing transgenic lines as compared to 0.07 mg g21 DW in
control tobacco plants (Fig. 5B). Similarly, EC content was also
increased and found in the range of 0.25–0.98 mg g21 DW in
CsDFR and CsANR overexpressing transgenic lines as compared to
0.167 mg g21 DW in control tobacco plants (Fig. 5C). EGC
content was observed in range of 9.4–21.5 mg g21 DW in CsDFR
and CsANR overexpressing transgenic lines as compared to
0.22 mg g21 DW of control tobacco plants (Fig. 5D). Spectra of
the HPLC runs, indicating identified peaks for Cat, EC and EGC
and their respective standards are shown in Figure S2. DMACA
staining of young leaf of CsDFR and CsANR overexpressing
transgenic tobacco also showed higher accumulation of flavan-3ols compared to control tobacco plants (Fig. S3).
CsDFR and CsANR cDNA in the genome of transgenic lines was
confirmed through PCR. In PCR, transgene specific primers were
used with total genomic DNA extracted from the mature leaves
and PCR products were confirmed by sequencing. A representative picture depicting the integration of CsDFR and CsANR cDNA
is shown only for three lines of each transgenic tobacco (Fig. 2B). A
specific band of 1.044 and 1.014 kbp has indicated the introduction of CsDFR and CsANR cDNA respectively in the transformed
tobacco lines. T2 homozygous lines of these transgenic tobaccos
were selected and confirmed three lines were further used for
CsDFR and CsANR expression analysis.
For CsDFR and CsANR transcript expression analysis, total RNA
was isolated from leaves of confirmed transgenic lines and used
forcDNA synthesis. A primer set specific to CsDFR and CsANR
cDNA was used for PCR amplification. Detection of CsDFR and
CsANR transcripts in transgenic lines has suggested the constitutive
expression of transgenes. While in control tobacco plants, no
expression was observed for the transgenes (Fig. 2C). Three CsDFR
overexpressing transgenic lines as D-01, D-15 and D-17 and three
CsANR overexpressing transgenic lines as A-05, A-08 and A-27
showed relatively better expression of CsDFR and CsANR
transcripts, respectively. Therefore, these lines were used for
various analyses.
Overexpression of CsDFR and CsANR enhanced
antioxidant potential and provided better resistance to
oxidative damage in transgenic tobacco
Tobacco plants overexpressing CsDFR and CsANR
showed early flowering and significantly higher yield
To check antioxidant potential, ability of the plant extract of
CsDFR and CsANR overexpressing transgenic lines and control
tobacco to scavenge a stable free radical DPPH was measured.
Methanolic extract of the leaves of CsDFR and CsANR overexpressing transgenic lines showed higher free radical scavenging
activity for DPPH by 70–185% as compared to control tobacco
plants (Fig. 6A). Result suggested the improved antioxidant
potential of transgenic tobacco upon CsDFR and CsANR overexpression.
Since CsDFR and CsANR overexpressing transgenic lines
showed higher levels of flavonoids and enhanced antioxidant
potential, the cellular response of transgenic lines to oxidative
damage was analyzed by determining ROS-induced cell death.
The CsDFR and CsANR overexpressing transgenic lines showed
apparent tolerance to oxidative damage monitored in the form of
H2O2 induced cell death. The H2O2 induced cell death rate was
lesser by 15–43% in CsDFR and CsANR overexpressing transgenic
lines as compared to control tobacco plants.
Transgenic lines vis-à-vis control plants were morphologically
characterized for flowering time. The relative growth of transgenic
lines overexpressing CsDFR and CsANR compared with the control
is shown in Fig. 3A, B. The CsDFR and CsANR overexpressing
transgenic lines were flowered early and completed their life cycle
10–15 days in advance compared to control. Close up views
showing the phenotype of early flowering in CsDFR and CsANR
overexpressing transgenic tobacco lines (Fig. 3C). Transgenic
tobacco lines overexpressing CsDFR (D) and CsANR (A) flowered
after an average of 86 days (D-01), 95 days (D-15), 86 days (D-17),
90 days (A-05), 88 days (A-08), and 85 (A-27) days of seed
germination compared to 105 days to flower after seed germination in control tobacco plants (Fig. 3D). The number of capsules
and seed yield per plant was improved in CsDFR and CsANR
transgenic lines as compared to control tobacco plants (Fig. 3D).
The capsule yield was not uniformly improved. However, there
was an uniform improvement in seed yield per capsule of
transgenic lines as compared to control tobacco plants (Fig. 3 E,
F). The weight of thousand seeds in transgenic lines was found to
be higher than control tobacco plants (Fig. 3G).
Transgenic lines exhibited anti-herbivores effect
To see the anti-herbivores effect of CsDFR and CsANR
overexpression in tobacco, leaf discs of transgenic lines were
exposed to one-day-old S. litura larvae. On third day, there was
visible difference of feeding by S. litura on leaf discs of transgenic
tobacco and control tobacco. The leaf discs of CsDFR and CsANR
transgenic lines showed lowest feeding by S. litura as compared to
control leaf discs (Fig. 7A, B). In other words, leaf discs of CsDFR
and CsANR overexpressing transgenic tobacco showed stronger
negative effect on growth and survival of S. litura larvae relative to
leaf discs of control. This response of transgenics could be due to
toxic effects of higher accumulated flavonoids to S. litura larvae.
Hence, the observed negative effect on the larvae was because they
could not eat transgenic leaves and died because of starvation.
This was particularly evident in all lines of CsDFR and CsANR
overexpressing transgenic tobacco. Percentage growth inhibition
of larvae feeding on leaves of D-01, D-15 and D-17 was 10%, 36%
and 20% respectively relative to the growth of larvae feeding on
control leaf discs (Fig. 7C). Similar results were observed for
CsANR overexpressing transgenic lines. Percentage growth inhibi-
Overexpression of CsDFR and CsANR increased NtCHS,
NtANR2 transcript expression and flavonoids
accumulation in transgenic tobacco
Interestingly, CsDFR and CsANR overexpressing transgenic lines
showed upregulation in transcript expression levels of native
tobacco NtCHS and NtANR2 genes (Fig. 4A–D). While there was
no significant change in the transcript levels of various other native
tobacco NtPAL, NtC4H, Nt4CL, NtCHI, NtF3H, NtDFR, NtFLS,
NtANR1 and NtANS genes of flavonoid pathway in both CsDFR
and CsANR overexpressing transgenic lines (Fig. S1).
The relative amount of total flavonoids, and flavan-3-ols (Cat,
EC, and EGC) was determined in the transgenic lines vis-à-vis
control tobacco plants. Total flavonoids content of control tobacco
was 39.8 mg quercetin equivalent (QE) g21 DW. While, the total
flavonoids content was estimated higher in the range of 47.63–
69.77 mg QE g21 DW in CsDFR and CsANR overexpressing
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Figure 3. Morphological characterization and yield parameters of CsDFR and CsANR overexpressing transgenic lines vis-à-vis mock
tobacco plants. CsDFR and CsANR overexpressing transgenic lines were longer than mock tobacco plants (A, B). Close up views showing early
flowering in CsDFR and CsANR transgenic lines compared to mock tobacco (C). The CsDFR and CsANR overexpressing transgenic lines produced early
flowering as compared to control tobacco plants (D). The capsule yield (E), seed yield (F) and seed weight per thousand seed (G) in CsDFR and CsANR
overexpressing transgenic lines as compared to mock tobacco plants. Mean 6 SD from three replications are shown. Statistical significance is
indicated as (*) for P,0.05.
doi:10.1371/journal.pone.0065535.g003
peaks for these two phytohormones and standard are shown in
Figure S4.
tion of larvae feeding on leaves of A-05, A-08 and A-27 was 70%,
15% and 40% respectively relative to growth of larvae feeding on
control leaf discs (Fig. 7D). The lower survivorship and slower
growth of larvae on CsDFR and CsANR overexpressing transgenic
lines has indicated the reduced vigor of S. litura larvae.
Discussion
Flavonoids are ubiquitous in their distribution and are
important for various fundamental functions in plants. They act
as phytoalexins against pathogen and predators and impart
astringency to plants making them unpalatable [37,38]. They also
increase antioxidant potential, provide protection against stresses
and increase agronomical, economical and medicinal value of
several plants [1,5,38]. Flaovnoids in the dietary source provide a
major source of antioxidants to combat diseases and improvement
of human health [3,5]. Extraordinary high content of flavonoids in
tea plant has encouraged us to investigate its flavonoid pathway.
CsF3H, CsDFR and CsANR encoding enzymes have been identified
as important for flavonoids biosynthesis in tea [10,17,27]. Among
them, CsDFR and CsANR encoding enzymes are known to catalyze
stereo-specific reduction of substrate using NADPH as a cofactor.
Therefore, in this study transgenic tobacco overexpressing cDNA
from tea encoding DFR and ANR enzymatic proteins were
Transgenic lines show modulation in endogenous GA3
and IAA levels
To check the influence of CsDFR and CsANR overexpression on
phytohormone levels, the endogenous GA3 and IAA levels were
measured by HPLC. The GA3 level was found to be decreased in
transgenic tobacco lines as compared to control tobacco plants.
GA3 level was observed in the range of 49–110.3 ng/g FW in
CsDFR and CsANR overexpressing transgenic lines compared to
138.80 ng/g FW in control tobacco plants (Fig. 8A). On contrary,
the IAA level was found to be higher in transgenic lines as
compared to control tobacco plants. IAA level was observed in the
range of 810.5–1010.8 ng/g FW in CsDFR and CsANR overexpressing transgenic lines as compared to 780.2 ng/g FW in control
tobacco plants (Fig. 8B).The spectra of HPLC runs with identified
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Figure 4. Transcript expression level of NtCHS and NtANR2 gene enhanced in CsDFR and CsANR transgenic tobacco. A, Transcript
expression level of NtCHS in CsDFR transgenic lines. B, Transcript expression level of NtCHS in CsANR transgenic lines. C, Transcript expression level of
NtANR2 in CsDFR transgenic lines. D, Transcript expression level of NtANR2 in CsANR transgenic lines. Expression of 26S rRNA was used as internal
control and experiment was repeated at least three times. Below gel pictures relative level of expression is shown with bar diagram and values are
mean of three replications with error bars indicating 6 SD. Statistical significance is indicated as (*) for P,0.05.
doi:10.1371/journal.pone.0065535.g004
hormones are known to affect the accumulation of secondary
metabolites and vice versa [30,39,40]. In water deficit condition,
the accumulated flavonoids content was observed to decrease
endogenous GA3 level and increase endogenous IAA level in
leaves of Scutellaria baicalensis [40]. The decrease in GA3 level and
increase in endogenous IAA level was also observed in transgenic
tobacco overexpressing CsDFR and CsANR. High level of GA3
inhibited the flowering in citrus plants [41]. While topical
application of IAA has induced flowering in Arabidopsis [42].
Thus, the modulation of endogenous GA3 and free IAA level in
CsANR and CsDFR transgenic tobacco lines could be possible
reason for early flowering, improved fruit number, seed yield and
thousand seed weight.
Altered flavonoids content has been reported through metabolic
engineering of flavonoid pathway genes in several transgenic
plants [20,23,25,31,43]. Transgenic tobacco overexpressing
CsDFR and CsANR cDNA encoding for enzymatic proteins
showed higher contents of total flavonoids than control tobacco
plants. Also monomeric flavan-3-ols such as Cat, EC and EGC
were accumulated to higher extent in both CsDFR and CsANR
generated. These transgenic tobaccos were analyzed to see the
influence of overexpression of CsDFR and CsANR transgene onto
flavonoids accumulation, developmental and protective abilities
against biotic stress. CsDFR and CsANR overexpressing transgenic
tobacco lines have produced 10–15 days early flowering, better
capsules and thousand seed weight than control tobacco plants.
However, the mutants compromised at different steps in the
flavonoid biosynthetic pathway have been reported for affect on
shoot/flower number, overall architecture and stature, and
thousand seed weight [39]. Similarly, FLS silenced transgenic
tobaccos were reported with lesser height, delayed flowering and
lower yield than control tobacco plant [31]. The present study on
overexpression of CsDFR and CsANR in tobacco has reaffirmed the
link between flavonoids and reproductive phenotype by displaying
early flowering, improved fruit number, seed yield and thousand
seed weight. However, the mechanisms for these changes are not
well understood. The possible mechanisms could be either direct
interaction of flavonoids with unidentified molecular targets or
indirect effects mediated by the ability of flavonoids to modulate
the levels of GA and IAA or through ROS regulation [39]. Plant
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CsANR and CsDFR Transgenic Tobacco
Figure 6. CsDFR and CsANR overexpression increased free
radicals scavenging activity and lowered oxidative stress
induced cell death. A, The scavenging activity measured by DPPH
method in CsDFR and CsANR overexpressing transgenic lines. B, H2O2
induced cell death in CsDFR and CsANR overexpressing transgenic lines.
Data is the mean of three replications with error bars indicating 6 SD.
Statistical significance is indicated as (*) for P,0.05.
doi:10.1371/journal.pone.0065535.g006
enzymatic steps starting with chalcone synthase (CHS) that
catalyzes the condensation of 4-coumaroyl-CoA and malonylCoAs. CHS catalyzes the first regulatory step and regulates the
diversion of carbon flux towards flavonoid biosynthesis pathway.
Silencing and overexpression of CHS encoding gene has resulted
in a successful alternation of flavonoids in petunia and tomato,
respectively [44,45]. The silencing and overexpression of ANR
genes (ANR1 and ANR2) have also redirected the metabolic flux
either towards anthocyanins or PAs biosynthesis [20,46]. The
expression of many of genes encoding flavonoid pathway enzymes
like CHS, F3H, ANS and ANR is controlled by regulatory genes
such as PAR (proanthocyanidin regulator). Hence, the overexpression of CsDFR and CsANR might have affected the expression of
selected endogenous genes through influencing the regulators or
only the genes encoding endogenous regulatory enzymes of
flavonoid pathway showed influence at expression level. Further,
CsDFR and CsANR were continuously expressed by a constitutive
promoter 35S and need continuous substrate in transgenic
tobacco. This might be avoiding the feedback inhibition of their
expression which is otherwise natural possible situation. The
upregulation of transcript level of NtCHS and NtANR2 in
transgenic tobacco overexpressing CsDFR and CsANR might
further aid to the accumulation of total flavonoids including PAs
and monomeric flavan-3-ols.
The accumulation of flavan-3-ols in CsDFR and CsANR
overexpressing transgenic lines was histochemically confirmed by
using DMACA staining. The elevation in EC and GC (gallocatechin) content has also been reported earlier in tobacco coexpressing PAP1 and MtANR [19]. However, the EC and GC
content in individually overexpressing MtANR and PAP1 tobacco
Figure 5. The CsDFR and CsANR overexpressing transgenic lines
show higher contents of total flavonoids and increased flavan3-ols content in transgenic tobacco compared to control
tobaccos. Three flavan-3-ols namely catechin, epicatechin and
epigallocatechin were measured in CsDFR and CsANR transgenic lines
vis-à-vis control tobacco. Total flavonoids in CsDFR and CsANR
overexpressing transgenic tobaccos (A). The catechin (B), epicatechin
(C) and epigallocatechin (D) contents in CsDFR and CsANR transgenic
lines as well as control tobacco plants. Data is the mean of three
replications with error bars indicating 6 SD. Statistical significance is
indicated as (*) for P,0.05.
doi:10.1371/journal.pone.0065535.g005
overexpressing transgenic tobacco than control plant. Importantly,
overexpression of CsDFR and CsANR cDNA in transgenic tobacco
has also upregulated the expression of NtCHS and NtANR2 gene,
while other genes encoding enzymes of flavonoid pathway
remained unaffected. Flavonoids are synthesized in a series of
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CsANR and CsDFR Transgenic Tobacco
Figure 7. CsDFR and CsANR overexpression provided anti-herbivores effect against S. litura in transgenic tobacco. The picture showed
less feeding by S. litura on leaf discs of CsDFR transgenic tobacco line (A) and CsANR transgenic tobacco line (B) as compared to leaf discs of control
tobacco plants. The relative percentage growth inhibition of S. litura feeding on leaf discs of selected CsDFR lines (C) and CsANR lines (D) as compared
to relative percentage growth inhibition on leaf discs of control tobacco plants. Data is the mean of three replications with error bars indicating 6 SD.
doi:10.1371/journal.pone.0065535.g007
activities than control tobacco plants. Furthermore, the improved
total free radical scavenging activity has encouraged us to check
the tolerance of plant against oxidative stress. The overexpression
of CsDFR and CsANR in transgenic lines has lowered H2O2
mediated cell death, confirming their tolerance against oxidative
stress. Similarly, the overexpression of CsDFR in transgenic rice
has also been reported to provide cell death tolerance [33].
Level of phenolic compounds, especially flavonoids in leaf
extracts of various plant species have been documented for plant
anti-herbivore defense [37]. Under this category, transgenic
tobaccos accumulating caffeine (alkaloids) and rutin (flavonol)
have been reported to provide resistance against S. litura [34,47].
Similarly, elevation in the contents of flavan-3-ols and PAs has
plants was not affected [19]. Our recent study has also
documented the elevation in Cat, EC and EGC levels upon FLS
silencing in tobacco plants. The FLS silencing has inhibited the
production of flavonols and directed the carbon flux towards
biosynthesis of Cat, EC and EGC [31].
Flavan-3-ols act as antioxidant by scavenging free radicals as
well as by breaking the free radical chain reactions [5,38]. High
antioxidant potential and free radical scavenging activity of tea has
been documented due to its higher levels of polyphenols especially
flavan-3-ols [32]. Since CsDFR and CsANR overexpressing
transgenic lines showed higher levels of flavonoids, they were
evaluated for their free radical scavenging activity. Both types of
transgenics have shown higher total free radical scavenging
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CsANR and CsDFR Transgenic Tobacco
In conclusion, overexpression of tea cDNA CsDFR and CsANR
encoding DFR and ANR enzymatic proteins in transgenic tobacco
has induced early flowering and improved seed yield. The CsDFR/
CsANR overexpression has increased the accumulation of flavonoids, thereby improved antioxidant potential and redox state of
transgenic tobacco plants. Ultimately, improved antioxidant
potential upon CsDFR and CsANR overexpression in transgenic
tobacco has provided the biotic stress tolerance against S. litura.
Supporting Information
Figure S1 Transcript expression analysis of genes
encoding various enzymes of flavonoid biosynthetic
pathway in CsDFR and CsANR overexpressing transgenic lines as well as control tobacco plants.
(TIF)
Figure S2 HPLC spectra of standard catechin (Cat),
epicatechin (EC) and epigallocatechin (EGC) are on the
left hand side. On the right hand side, peaks are identified for
Cat, EC and EGC from the extracts of transgenic as well as
control tobacco plants by comparing with their respective
standard.
(TIF)
Figure S3 DMACA stained leaf of CsDFR and CsANR
overexpressing transgenic tobacco plant vis-à-vis control tobacco plants.
(TIF)
Figure 8. Modulation in phytohormones level in CsDFR and
CsANR overexpressing transgenic lines. Endogenous GA3 was
decreased (A) and free indole acetic acid (IAA) was increased (B) in
transgenics. Data is the mean of three replications with error bars
indicating 6 SD. Statistical significance is indicated as (*) for P,0.05.
doi:10.1371/journal.pone.0065535.g008
HPLC spectra of standard gibberellin (GA3)
and indole-3-acetic acid (IAA) are on the left hand side.
On the right hand side, peaks for GA3 and IAA were identified
from the extracts of transgenic as well as control tobacco plants by
comparing with their respective standard.
(TIF)
Figure S4
been reported in response to Vaccinium myrtillus infection [48]. The
flavonoids have also been reported as insecticidal to Helicoverpa zea
[4]. In insect gut, these compounds are known to facilitate the
conversion of amino acids to more toxic quinones. Such toxic
quinones have been reported to reduce the availability of free
amino acid and protein by binding to –SH and –NH2 groups
[4,39]. There was a strong negative effect on growth and survival
of S. litura upon feeding on leaves of CsDFR and CsANR
overexpressing transgenic tobacco. Higher levels of monomeric
and polymeric flavan-3-ols could be responsible for such an antiherbivores activity. Their higher content in transgenic tobacco
might be acting as anti-nutrient and therefore, reduced the
digestibility of protein by S. litura either by precipitation of protein
or by inhibiting the enzyme activity [4,5]. These phytochemicals
also impart astringency that avoids plant feeding by herbivores [4–
6]. Thus, higher levels of flavonoids in CsDFR and CsANR
overexpressing transgenic tobacco could be the possible reason for
providing protection against feeding by herbivores S. litura.
Table S1 List of primers used for expression analysis of
various genes encoding enzymes of flavonoid biosynthetic pathway.
(TIF)
Acknowledgments
We are grateful to the Director, CSIR-IHBT for encouragement and
suggestions. We would like to thank Dr. Ashu Gulati and Mr. Robin Joshi
for their help in estimation of flavonoids by HPLC method. VK is also
thankful to CSIR for providing research fellowship in the form of SRF.
Author Contributions
Conceived and designed the experiments: VK GN SK SKY. Performed
the experiments: VK GN. Analyzed the data: VK SKY. Wrote the paper:
VK SKY.
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