Plant Biotechnology Journal (2014) 12, pp.

6980

doi: 10.1111/pbi.12118

Co-expression of Arabidopsis transcription factor,

AtMYB12, and soybean isoflavone synthase, GmIFS1,
genes in tobacco leads to enhanced biosynthesis of
isoflavones and flavonols resulting in osteoprotective
activity
Ashutosh Pandey1,, Prashant Misra1,,, Mohd P. Khan2, Gaurav Swarnkar2, Mahesh C. Tewari2, Sweta
Bhambhani1, Ritu Trivedi2, Naibedya Chattopadhyay2 and Prabodh K. Trivedi1,*
1

Council of Scientific and Industrial Research-National Botanical Research Institute (CSIR-NBRI), Lucknow, India

CSIR-Central Drug Research Institute (CSIR-CDRI), Endocrinology Division and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI),

Jankipuram Extension, Lucknow, India

Received 14 May 2013;

accepted 9 August 2013.
*Correspondence (Tel +91-522-2297958;
fax +91-522-2205836, 2205839;
e-mail prabodht@hotmail.com,
prabodht@nbri.res.in)

These authors contributed equally to this

study.

Present address: CSIR-Indian Institute of

Integrative Medicine (IIIM), Canal Road,
Jammu 180001, India.

Keywords: AtMYB12, GmIFS1,

isoflavone, metabolic engineering,
osteoporosis, transgenic plant.

Summary
Isoflavones, a group of flavonoids, restricted almost exclusively to family Leguminosae are known
to exhibit anticancerous and anti-osteoporotic activities in animal systems and have been a target
for metabolic engineering in commonly consumed food crops. Earlier efforts based on the
expression of legume isoflavone synthase (IFS) genes in nonlegume plant species led to the
limited success in terms of isoflavone content in transgenic tissue due to the limitation of
substrate for IFS enzyme. In this work to overcome this limitation, the activation of multiple
genes of flavonoid pathway using Arabidopsis transcription factor AtMYB12 has been carried
out. We developed transgenic tobacco lines constitutively co-expressing AtMYB12 and GmIFS1
(soybean IFS) genes or independently and carried out their phytochemical and molecular
analyses. The leaves of co-expressing transgenic lines were found to have elevated flavonol
content along with the accumulation of substantial amount of genistein glycoconjugates being
at the highest levels that could be engineered in tobacco leaves till date. Oestrogen-deficient
(ovariectomized, Ovx) mice fed with leaf extract from transgenic plant co-expressing AtMYB12
and GmIFS1 but not wild-type extract exhibited significant conservation of trabecular
microarchitecture, reduced osteoclast number and expression of osteoclastogenic genes, higher
total serum antioxidant levels and increased uterine oestrogenicity compared with Ovx mice
treated with vehicle (control). The skeletal effect of the transgenic extract was comparable to
oestrogen-treated Ovx mice. Together, our results establish an efficient strategy for successful
pathway engineering of isoflavones and other flavonoids in crop plants and provide a direct
evidence of improved osteoprotective effect of transgenic plant extract.

Introduction
Flavonoids synthesized by phenylpropanoid pathway are ubiquitous in distribution between different plant species. These
molecules participate in a myriad of physiological and biochemical
processes in plants and have been studied extensively for their
effects on human health (Ververidis et al., 2007; Winkel-Shirley,
2001). On the basis of chemical structure, flavonoids have been
divided into different classes, such as flavanones, flavonols,
isoflavones and flavones. The activities and health-promoting
effects of different flavonoids differ depending on the association
with specific class. The biosynthesis of certain flavonoids is
restricted to a specific taxonomic group(s) due to the presence or
absence of specific enzymes involved in the biosynthesis.
Isoflavones represent such a subgroup of flavonoids that are
almost exclusively restricted to the family Leguminosae of plant
kingdom (Figure 1a, Veitch, 2007). In legumes, isoflavonoids
have been implicated in plantmicrobe interaction, inducer of

Nod gene of nitrogen fixing bacteria (Van Rhijn and Vanderleyden, 1995; Pueppke, 1996, Dixon, 1999; Subramanian et al.,
2007). Biosynthesis of these molecules is known to be induced by
defence signal elicitors such as jasmonic acid and salicylic acid
(Dixon, 1999; Subramanian et al., 2007; Misra et al., 2010a).
Simple isoflavones such as daidzein and genistein harbour
antimicrobial activity and are also precursor of complex
isoflavonoids, phytoalexins (Samac and Graham, 2007). In
addition to benefits to plant itself, these molecules have been
shown to improve human health through protecting against
hormone-dependent cancers, cardiovascular disease, osteoporosis and menopausal symptoms (Cornwell et al., 2004; Dixon,
2004; Srivastava et al., 2013a,b; Tyagi et al., 2010; Pandey et al.,
2010; Trivedi et al., 2009). Based on the in vitro, in vivo and
epidemiological studies, isoflavonoids have been reported to
exhibit antioxidant, antimutagenic, anticarcinogenic, anti-osteoporotic and antiproliferative activities in human and animal
systems (Birt et al., 2001; Iwasaki et al., 2008; Miadokova et al.,

2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

69

70 Ashutosh Pandey et al.

(a)

(b)

Figure 1 Phenylpropanoid pathway and constructs used for plant

transformation. (a) Schematic representation of general phenylpropanoid
pathway with flavonoid pathway showing biosynthesis of aglycone
backbones of isoflavones and flavonols. Steps shown in green are almost
exclusively present in legume taxa. The genes encoding the enzymes
shown in red are up-regulated by AtMYB12 transcription factor in
tobacco. (b) Schematic representation of T-DNA region of plant expression
constructs carrying AtMYB12 and GmIFS1 genes in pBI121 vector used for
tobacco transformation.

2002; Miadokova, 2009; Ryan-Borchers et al., 2006; Scarpato

et al., 2008).
The health-beneficial properties of isoflavones are primarily
attributed to their structural similarity to the mammalian
hormone oestrogen, and therefore, a term phyto-oestrogen
is often ascribed to these compounds (Dixon and Ferreira,
2002). Soybean is the major source of dietary isoflavones such
as daidzein and genistein. Epidemiological studies have suggested that the daily intake of soybean rich diet leads to
protection against postmenopausal osteoporosis as well as
certain hormone-dependent cancers due to the presence of
isoflavones (Miadokova, 2009). Owing to such health-beneficial
properties of isoflavones, it is desirable to incorporate or
improve their biosynthesis in commonly consumed nonleguminous crops, fruits and vegetables through transgenesis (Misra
et al., 2010a; Ogo et al., 2013).
Isoflavone biosynthesis is well characterized, and the first and
entry step reaction for this group is catalysed by isoflavone
synthase (IFS), a cytochrome p450 monooxygenase (Fig. 1a,
Dixon, 1999; Jung et al., 2000; Steele et al., 1999). Naringenin
and liquiritigenin, flavanones and central precursor of most
flavonoids, serve as substrate for IFS enzyme leading to the
biosynthesis of genistein and daidzein, respectively. These aglycone isoflavones are often conjugated with glycosyl and malonyl
residues by glycosyl- and malonyltransferases enzymes (Dhaubhadel et al., 2008; Suzuki et al., 2007). In legumes, the isoflavo-

noid pathway is further elaborated by the presence of several

downstream steps that may lead to many complex isoflavonoids
acting as phytoalexins in biotic stress (Akashi et al., 2000, 2005;
Farag et al., 2008).
Naringenin is ubiquitously distributed in both legume and
nonlegume plant species and can be routed towards genistein
biosynthesis in nonlegumes by the expression of legume IFS.
Based on this fact, IFS genes have been expressed in nonleguminous plants such as Arabidopsis, tobacco, Petunia, lettuce and
tomato (Jung et al., 2000; Liu et al., 2002; Liu et al., 2007; Misra
et al., 2010a; Shih et al., 2008; Tian and Dixon, 2006; Yu et al.,
2000). However, in all these efforts, the accumulation of
genistein in transgenic plants remained poor and restricted to
the tissues where flavonoid pathway activity was sufficient
enough to deliver substrate flux to IFS enzyme. Therefore, apart
from the expression of IFS gene, it was realized to activate the
flavonoid pathway for the metabolic engineering of isoflavones.
Tian and Dixon (2006) constructed bifunctional isoflavone
synthase/chalcone isomerase (IFS/CHI) enzyme and expressed in
tobacco but did not achieve enhanced level of isoflavones in leaf
tissue. Recently, Ogo et al. (2013) expressed PAL, CHS and
GmIFS1 together in rice and achieved enhanced genistein
accumulation in rice grain. This suggests that limiting substrate
is the bottleneck for the synthesis of isoflavones in desired tissues
of nonleguminous plants. In soybean, a single repeat MYB
protein, GmMYB176, has been implicated in the regulation of the
isoflavone biosynthesis through targeting the promoter of CHS8
gene (Yi et al., 2010). However, overexpression of GmMYB176 in
hairy roots of soybean did not result in the elevation in isoflavone
content as well as CHS8 expression, suggesting involvement of
additional factors to enhanced biosynthesis of isoflavones.
The transcription factors can regulate several steps in the
flavonoid biosynthetic pathway by targeting multiple structural
genes simultaneously (Grotewold, 2008). In our earlier study
(Misra et al., 2010b), we demonstrated that the expression of
Arabidopsis transcription factor, AtMYB12, in tobacco up-regulated phenylpropanoid pathway genes as well as other pathways
to enhance flux availability for flavonoid biosynthesis in general
and flavonol biosynthesis in particular. In the present study, we
have utilized AtMYB12 transcription factor for enhancing flux for
metabolic engineering of isoflavones in nonleguminous model
plant, tobacco. Our results suggest that the simultaneous
co-expression of AtMYB12 and GmIFS1 in tobacco leads to the
accumulation of substantial amount of genistein, not reported as
yet in any transgenic plants developed, in addition to flavonols.
We further demonstrate that daily administration of leaf extract
co-expressing transgenic tobacco lines to ovariectomized mice
confers improved bone conservation by retarding the activity of
osteoclasts in a manner similar to oestrogen. We suggest that
AtMYB12 in combination with other enzymes involved in
secondary conversion of flavonoid backbone can be used for
metabolic engineering of specific flavonoids in different plants
without using several structural genes for improved human
health, particularly bone health.

Results
Development of transgenic plants expressing different
genes
We developed three constructs, for developing transgenic tobacco
plants. CaMV-IFS and CaMV-MYB12 constructs were prepared to
develop GmIFS1- and AtMYB12-expressing transgenic lines.

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Isoflavone metabolic engineering and improved bone health 71

CaMV-MYB12 + IFS construct was developed to co-express both
the genes in transgenic lines. A co-expression construct allowing
simultaneous constitutive expression of AtMYB12 and GmIFS1
genes was developed in plant expression vector pBI121 by placing
both the expression cassettes at same T-DNA region (Figure 1b).
Constitutive CaMV35S promoter was used to express these genes
for studying limitation of substrate flux in leaf and flower tissue in
same transgenic line. The Agrobacterium-mediated tobacco transformation was carried out, and several independent kanamycinresistant transgenic tobacco lines were generated for each
construct. In all the transgenic lines, no phenotypic change or
growth penalty was observed in comparison with WT plants.
However, transgenic plants transformed with AtMYB12 or
AtMYB12 + GmIFS exhibited depletion in petal pigmentation
(Figure 2a), a phenotypic characteristic of AtMYB12-expressing
transgenic tobacco lines (Luo et al., 2008; Misra et al., 2010b). The
transgenic lines developed in the study were confirmed through
genomic DNA and RT-PCR. Genomic DNA amplification from the
DNA isolated from AtMYB12 + GmIFS showed presence of both
the genes (Figure 2b). The transgenic lines developed after
transformation with the constructs having AtMYB12 and GmIFS1
alone showed the presence of respective genes only. RT-PCR
analysis of selected transgenic lines suggested high-level expression
of AtMYB12 and GmIFS1 in the co-expressing lines (Figure 2c).

Modulated expression of genes involved in flavonoid

biosynthesis in transgenic lines
To study the expression of flavonoid biosynthesis genes, the
quantitative real-time PCR was performed using RNA from leaves
and flower petals of different transgenic lines and WT tobacco
plants. Three independent AtMYB12- and GmIFS-co-expressing

as well as AtMYB12 and GmIFS, transgenic lines were taken for

the study. The expression of various key genes of phenylpropanoid pathway and flavonoid pathway such as PAL, CHI, CHS, F3H
and FLS was up-regulated several folds in co-expressing and
AtMYB12-expressing transgenic tobacco plants in comparison
with WT and GmIFS1 transgenics (Figure 3). However, enhancement in the expression of the genes was much more in leaf in
comparison with petals. These results establish that the expression of AtMYB12 in AtMYB12 and GmIFS1 co-expressing or
AtMYB12 alone has enhanced the expression of pathway-related
genes that might have modulated different pathways to enhance
flux in phenylpropanoid pathway leading to enhanced flavonol
content. No such enhanced expression of pathway-related genes
was observed in transgenic lines expressing GmIFS1.

Phytochemical analysis of transgenic tobacco plants

The methanolic and acid-hydrolysed methanolic extracts from the
leaves of transgenic as well as WT plants were analysed for
flavonoid quantification through HPLC. The analysis of unhydrolysed methanolic extracts suggested rutin as the most abundant
flavonol in leaves of various transgenic lines and WT (Figure S1).
Rutin contents in leaves of AtMYB12- and AtMYB12- and
GmIFS1-co-expressing (up to 2.8  0.32 mg/g FW) transgenic
tobacco lines were several fold higher as compared to the leaves
from
WT
and
GmIFS1-transgenic
tobacco
plants
(0.055  0.0072 mg/g FW). The methanolic extracts were acidhydrolysed to release various aglycone forms of flavonoid and
analysed after HPLC separation (Figure S2). In acid-hydrolysed
extracts, apart from various flavonols (quercetin and kaempferol),
genistein (up to 0.058  0.007 mg/g FW) was present in leaves
of co-expressing transgenic plants (Figure 4). The identity of

(a)

(b)

Figure 2 Analysis of transgenic lines for flower

colour and expression of transgenes. (a) Flower
colour alterations in different transgenic lines. The
pigmentation in petals of co-expressing lines
(AtMYB12 + GmIFS) was compared with WT,
AtMYB12- and GmIFS1-expressing tobacco
transgenic lines. (b) Confirmation of the presence
of the transgene by PCR amplification of
transgenes using CaMV 35S forward and NosT
reverse primers in different transgenic lines. (c)
Expression analysis of transgenes through semiquantitative RT-PCR using total RNA from leaves
of different transgenic lines.

(c)

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72 Ashutosh Pandey et al.

Figure 3 Modulation of expression of flavonoid

biosynthesis-related genes. Quantitative
expression analysis of the genes involved in
phenylpropanoid pathway in different transgenic
lines. Expression of structural genes of
phenylpropanoid pathway/flavonoid pathway was
analysed using RNA from the leaves and petals of
different transgenic lines. PAL, phenylalanine
ammonia lyase; FLS, flavonol synthase; CHI,
chalcone isomerase; CHS, chalcone synthase; F3H,
flavanone 3-hydroxylase.

genistein was confirmed using mass spectrometric analysis of

these samples (Figure S3). Genistein was not detected in leaf of
AtMYB12- or GmIFS1-alone- expressing transgenic lines. In
addition, the co-expressing transgenic lines accumulated higher
contents of quercetin and kaempferol (Figure 4), which was up to
30-folds of the flavonol content of the WT leaves. The flavonol
contents was not significantly different between AtMYB12-aloneor AtMYB12- and GmIFS1-co-expressing transgenic lines which
suggest that due to the presence of GmIFS1, substrate flux for
flavonol biosynthesis was not limiting. We also measured the
accumulation of different flavonoids in petals of different
transgenic lines. Petals of GmIFS1-expressing and AtMYB12and GmIFS1-co-expressing lines accumulated similar level of
genistein. This suggests that the expression of AtMYB12 could
provide sufficient flux in leaf tissue, comparable to flower, for the
synthesis of genistein. Together, these results demonstrate that
the combined expression of a flavonoid activator transcription
factor gene (AtMYB12) and IFS can allow the biosynthesis of
isoflavones in a tissue, which does not appear to have proper
amount of substrates for IFS.

Effects of leaf extracts on body weight, serum

antioxidant level and trabecular microarchitecture in
Ovx mice
Adult Balb/c mice, sham and ovariectomized (Ovx), were daily
administered leaf extract by gavage of wild type (designated as

WT50 and WT100, corresponding to 50 and 100 mg/kg doses)

and transgenic line co-expressing AtMYB12 and GmIFS1 (designated as T50 and T100, corresponding to 50 and 100 mg/kg
doses) for 4 weeks. Both treatments were generally well tolerated
for the duration (4 weeks) of administration. OVx resulted in
increased body weight compared with the ovary intact, sham
group. Body weight was not different between the 17b-estradiol
(E2) and sham groups. Treatment of Ovx mice with WT or
transgenic leaf extracts had no effect on the OVx-induced weight
gain (Figure 5a).
Because flavonoids have antioxidant property, we measured
total antioxidant levels in the serum of various groups at the end
of 4 weeks. In comparison with the sham, antioxidant level was
decreased in all Ovx groups except Ovx + E2 and Ovx + T100
where the levels were comparable (Figure 5b).
Histomorphometric parameters were analysed using lCT.
Reconstructed 3D-lCT images revealed deterioration of the
trabecular architecture due to destruction of trabecular bones
of excised femur in Ovx + vehicle group compared with numerous and well-connected trabeculae in sham + vehicle group,
suggesting significant induction of osteopenia due to Ovx
(Figure 5c). Trabecular response to treatment of Ovx mice by
WT and transgenic extracts was quantified at the femur epiphysis,
and the values were compared with that of sham, Ovx + vehicle
and Ovx + E2 groups. Data showed that compared with the
sham, the Ovx + vehicle group had reduced trabecular bone

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Isoflavone metabolic engineering and improved bone health 73

Figure 4 Flavonoid content in transgenic lines.

Phytochemical analysis of the hydrolysed
methanolic extracts of leaves and petals of
different transgenic lines. Compounds were
quantified by separating hydrolysed methanolic
extracts from the young leaves and petals of WT
and transgenic lines using HPLC. A typical HPLC
chromatogram is provided in Figure S2. The graph
shows values  SD of three leaves from each of
the independent transgenic line.

fraction (BV/TV), trabecular number (Tb.N), trabecular thickness

(Tb.Th) and connectivity density (Conn.D) and increased trabecular separation (Tb.sp) and degree of anisotropy (DA) (Figure 5d).
WT extract at both doses as well as the lower dose (50 mg/kg) of
transgenic extract administered to Ovx mice had all the lCT
parameters comparable to Ovx + vehicle group, suggesting their
failure to conserve Ovx-induced trabecular loss. However, at
100 mg/kg dose, transgenic extract administered to Ovx mice
completely preserved all the lCT parameters as the values were
comparable to the sham and Ovx + E2 groups.

Effects of leaf extracts on the formation of osteoclasts

from bone marrow precursors
At the end of various treatments to mice, we performed ex vivo
osteoclastogenesis by treating bone marrow cells (BMC) with
osteoclastogenic cytokines, RANKL (receptor activator of nuclear
factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) and examined the expression of major osteoclasts
differentiation markers including tartrate-resistant acid phosphatase 5b (TRAPC5b), and cathepsin-K (Botolin et al., 2005; Khan
et al., 2012). Figure 6 showed that compared with the sham
group, BMC response to osteoclastogenic stimulus in Ovx + vehicle
group was much greater as assessed by the expression of
TRAPC5b and cathepsin-K, suggesting increased osteoclast
differentiation from the precursor cells due to oestrogen deficiency. Levels of these two osteoclastogenic genes were different
between Ovx + vehicle and Ovx mice given WT extract at both
doses or the lower dose (50 mg/kg) of the transgenic extract,
suggesting that these treatments had no effects in altering
increased osteoclastogenic response induced by Ovx. At 100 mg/
kg dose, the transgenic-extract-treated Ovx mice strongly sup-

pressed ex vivo osteoclastogenesis evident from the levels of both

TRAPC5b and cathepsin-K being significantly lower than the
sham group but comparable to Ovx + E2 group (Figure 6).

Effects of leaf extract on osteoclasts in vivo

Histomorphometric analysis of the epiphysis in the proximal
femur showed that the number of osteoclasts (OC)/bone perimeter (N.Oc/B.Pm) and OC surface/bone surface (%) was significantly higher in the Ovx + vehicle group compared with the sham
(Table 1). These two parameters in the Ovx rats treated with WT
extracts were not different from Ovx + vehicle. These osteoclast
parameters were decreased (albeit nonsignificant) in transgenicextract-treated Ovx rats (Ovx + T50) compared with Ovx + vehicle. However, osteoclast number and surface in Ovx + T100 were
comparable to sham and Ovx + E2 groups (Table 1).

Effects of leaf extracts on oestrogenicity

As various flavonoids are known to have oestrogenic potency, we
studied uterine parameters at the termination of the treatments
shown in Table 2. Ovx resulted in reduced uterine weight
compared with sham group. E2 treatment to Ovx mice (positive
control) resulted in significant increase in uterine weight
compared with Ovx mice. Uterine weight was not different
between the Ovx + vehicle- and Ovx + WT extract-treated
groups. However, uterine weight of transgenic groups (at both
doses) was significantly higher than that of Ovx + vehicle group.
Analysis of uterine histomorphometry (representative photomicrograph of the uterine cross-section in various groups in
Figure S4) showed that Ovx resulted in marked reduction in total
uterine area, luminal area and luminal epithelial cell height
compared with the sham group (Table 2). E2 supplementation to

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74 Ashutosh Pandey et al.

(a)

(b)

(c)

(d)

Figure 5 Body weight, serum antioxidant levels and lCT of femur epiphysis of mice. (a) Body weight gain (%) at the end point, (b) total serum antioxidant
levels at the end point, (c) representative lCT images of femur epiphysis at the end point and (d) quantification of lCT parameters including BV/TV, Tb.Sp,
Tb.N, Tb.Th, Conn.D and D.A. All values are expressed as mean  SEM (n = 8 mice/group); *P < 0.05, *8P < 0.01, ***P < 0.001 compared with
sham + vehicle and #P < 0.05, ## P < 0.01, ###P < 0.001 compared with Ovx + vehicle.

Ovx mice resulted in significant increase in all three parameters

compared with Ovx + vehicle group. None of these parameters
were different between the Ovx + vehicle- and Ovx + WT
extract-treated groups. However, all of these parameters were
dose-dependently higher in Ovx mice treated with transgenic
extract when compared with Ovx + vehicle group, and at the
100 mg/kg dose, the parameters were comparable to Ovx + E2
group. Together, these data suggested that the leaf extract of
transgenic plant possesses significant oestrogenic activity at the
uterine level, whereas WT was devoid of such activity.

Discussion
Isoflavones that are known to be synthesized in the family
Leguminosae are known for their health-beneficial roles such as
protection against hormone-dependent cancers and osteoporosis
in human and animal systems (Occhiuto et al., 2007; Peterson
and Barnes, 1991). There have been efforts to engineer their
biosynthesis in nonleguminous plants, but very limited success
could be achieved (Jung et al., 2000; Liu et al., 2002; Yu et al.,
2000; Tian and Dixon, 2006; Liu et al., 2007; Misra et al., 2010a;
Shih et al., 2008). The availability of sufficient substrate flux in the
form of naringenin has been established to be a major constraint
for the engineering of substantial amounts of isoflavones. In
tobacco, the flavonoid pathway is differentially regulated in
different plant parts with its enhanced activity in petals as evident

by anthocyanin accumulation in this tissue (Misra et al., 2010b).

Conversely, leaves have lesser flavonoid pathway activity, and
therefore, the expression of legume IFS genes did not lead to the
accumulation of substantial amounts of genistein, and moreover,
in certain reports, no genistein could be detected (Tian and Dixon,
2006; Liu et al., 2007; Misra et al., 2010a). The MYB family of
transcription factors is known to activate flavonoid biosynthesis
by targeting and up-regulating genes encoding enzymes of the
pathway (Hichri et al., 2011; Lepiniec et al., 2006). The expression pattern of these transcription factors has been attributed to
the differential regulation of genes involved in flavonoid biosynthesis under developmental and environmental cues. It has been
established that AtMYB12 modulates genomewide expression
and metabolome in transgenic tobacco lines regulate phenylpropanoid pathway as well as other metabolic pathways leading
to increased flux availability to this pathway (Luo et al., 2008;
Misra et al., 2010b; Pandey et al., 2012a). This observation
encouraged us to utilize this transcription factor in context of
metabolic engineering of isoflavones in tobacco by constitutively
co-expressing AtMYB12 and GmIFS1 genes and thereby activating flavonoid pathway in the vicinity of IFS position. We herein
demonstrated that the co-expression of AtMYB12 and GmIFS1
genes in tobacco leads to the accumulation of substantial amount
of genistein glycoconjugates along with flavonols in tobacco
leaves. For the best of our knowledge, the genistein synthesized
in transgenic lines developed, in this study, is the highest amount

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Isoflavone metabolic engineering and improved bone health 75

of genistein till date that reported through pathway engineering
in leaves of nonleguminous transgenic plants. Interestingly, the
accumulated content is comparable to the amount of genistein
found in the petals of transgenic tobacco expressing legume IFS
genes (Figure 4). Recently, Ogo et al. (2013) used constructs

Figure 6 Transgenic extract diminishes ex vivo osteoclastogenesis in

mice. BMC from different experimental groups were seeded with RANKL
and MCSF for 7 days. qPCR data showing comparative expression levels of
TRAPC5b and cathepsin-K; mean  SEM of 3 independent experiments,
*P < 0.05, **P < 0.01, ***P < 0.001 compared with sham + vehicle and
#
P < 0.05, ##P < 0.01, ###P < 0.001 compared with Ovx + vehicle.

containing three genes (GmIFS1, PAL and CHS) of the pathway

and showed the accumulation of isoflavones in rice grain.
However, amount reported in the study is low to our study.
Expression of AtMYB12 might have modulated other metabolic
pathways in addition to phenylpropanoid pathway leading to
increased flux availability for isoflavonoid biosynthesis (Figure 3).
This fact accounts for the improved genistein accumulation in
tobacco leaves in co-expressing transgenic lines.
In addition, co-expressing lines accumulated flavonols to the
similar level as present in AtMYB12-expressing transgenic lines. In
tobacco, the flavonoid pathway is differentially regulated in
different plant parts with its enhanced activity in petals as evident
by anthocyanin accumulation. Therefore, fold expression of genes
is higher in leaves in comparison with petals (Figure 3). AtMYB12
is a flavonol-specific regulator and directs flux towards flavonol
biosynthesis. As the expression of DFR is not up-regulated by
AtMYB12, most of the flux is directed for the biosynthesis of
flavonols leading to reduced anthocyanin pigmentation in petals.
Both isoflavones and flavonols are derived from the same
substrate, and flavanone 3 hydroxylase (F3H) competes with IFS
for naringenin (Figure 1a). However, in an earlier work, the
suppression of F3H expression in IFS-expressing transgenic
tobacco did not result into any improvement in genistein content
of the leaves (Liu et al., 2007). Furthermore, genistein glycoconjugates accumulate at substantial amounts in leaves of
co-expressing transgenic lines, but their amounts are not comparable to their flavonol contents that are comparatively at much
higher levels (Figure 4). It indicates that in comparison with IFS,
F3H enzyme may be more efficient in consuming the naringenin
for flavonol biosynthesis. One of the reasons for this efficient
utilization of naringenin by F3H may be the efficient channelling
of naringenin towards this enzyme. We observed that leaf tissue
could accumulate similar amount of isoflavone as synthesized in
GmIFS1-expressing petals (Figure 4). This suggests that substrate
availability for IFS might not be major concern in co-expressing
transgenic lines. The enzymes of phenylpropanoid pathway and
flavonoid pathway have been suggested to be arranged in the
form of multi-enzyme complexes known as metabolome, for

Table 1 Histomorphometric analysis of femoral epiphysis

Parameters

Sham

Ovx + Veh

Ovx + WT50

Ovx + WT100

Ovx + T50

Ovx + T100

Ovx + E2

N.Oc/B.Pm

5.75  1.7a

13.5  3.4x

13.5  2.3x

13.7  3.0x

9.5  1.2z

5.25  2.2a

4.7  1.7a

7.8  1.3x

8.0  1.6x

OC Surface/bone

3.7  0.21a

8.2  0.68x

6.3  0.75z

3.8  0.64a

3.6  1.6a

surface (%)
P < 0.05, bP < 0.01, aP < 0.001 versus Ovx + Veh, zP < 0.05, yP < 0.01, xP < 0.001 versus Sham. Results are mean  SEM, n = 4. Control (WT50 and WT100) is at

50 or 100 mg/kg/day, Transgenic (T50 and T100) is at 50 or 100 mg/kg/day, E2 at 10 lg/kg/day.

Table 2 Uterine parameters

Sham

Ovx + Veh

Ovx + WT50

Ovx + WT100

Ovx + T50

Ovx + T100

Uterine weight (mg)

102.5  15.13a

24.0  15.13x

26.0  1.83x

24.2  4.05x

39.8  6.65x,c

79.1  6.60x,a

Total uterine area (lm2)

145.6  6.15a

77.8  4.93x

78.6  6.02x

78.9  5.07x

84.9  1.66x

98.0  1.63x,a

Parameter

Luminal area (lm2)

12.5  0.622a

7.3  0.32x

Luminal epithelium height (lm)

0.40  0.007

0.11  0.010

8.2  0.71x
x

0.11  0.003

9.6  0.70x,a
x

0.12  0.002

9.09  0.24a
0.20  0.003

Ovx + E2

12.2  1.09a
x,a

0.34  0.038

87.0  10.13y,a
100.3  3.63x,a
9.7  0.69x,a

x,a

0.26  0.053x,a

P < 0.05, bP < 0.01, aP < 0.001 versus Ovx, zP < 0.05, yP < 0.01, xP < 0.001 versus Sham. Results are mean _ SEM, n = 8 mice per group.

Control (WT50 and WT100) is at 50 or 100 mg/kg/day, Transgenic (T50 and T100) is at 50 or 100 mg/kg/dayKhan et al., 2012, E2 at 10 lg/kg/day.
2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 6980

76 Ashutosh Pandey et al.

efficient channelling of substrates (Winkel, 2004; Jorgensen
et al., 2005). It appears that heterologous expression of IFS in
tobacco flavonoid pathway do not fit well in this metabolome
presumably resulting into lesser efficiency of naringenin utilization
by IFS. In addition, although the product of IFS catalysed reaction,
the 2-hydroxyisoflavonone can be nonenzymatically converted
into their corresponding isoflavones (here in genistein), the
enzymatic conversion by HID (2-hydroxyisoflavanone dehydratase) may ensure the more enhanced and efficient production of
isoflavones (Shimamura et al., 2007). Therefore, HID could be
another target to be further studied in metabolic engineering of
isoflavones in the tobacco transgenics developed by us. Apart
from it, it would be interesting to study the effect of suppression
of F3H gene in these co-expressing transgenic lines in context of
genistein accumulation.
We made functional evaluation for enrichment of various
flavonoids in the transgenic lines co-expressing AtMYB12 and
GmIFS1 in comparison with WT extract in the mouse model of E2
deficiency-induced osteopenia. Postmenopausal bone loss is
achieved in rodents by bilateral Ovx resulting in rapid erosion of
trabecular bones (Boyd et al., 2006). Others and we have shown
that kaempferol, quercetin, rutin and genistein individually have
osteoprotective effect in various osteopenic animal models
(Gupta et al., 2013; Horcajada-Molteni et al., 2000; Kumar
et al., 2010, 2012; Pie et al., 2006; Trivedi et al., 2008; Wattel
et al., 2003). These flavonoids exert favourable skeletal effects by
pleiotropic action out of which oestrogen-like and antioxidant
effects are predominant. Whereas kaempferol, quercetin and
rutin (flavonols) are ubiquitously present in fruits and vegetables,
availability of genistein (an isoflavonoid) is restricted to leguminous plants (Justesen et al., 1998; Veitch, 2007). Engineering a
single plant source to obtain higher amounts of both flavonol and
isoflavonoid is therefore desirable as it could provide substantial
enhancement of osteoprotective efficacy of these different
flavonoid classes.
Our data showed that the leaf extract from transgenic plant
dose-dependently increased oestrogenicity and serum antioxidant
levels in Ovx mice, whilst WT extract had no effect. The most
reliable bioassay for oestrogenicity of a given agent is to study its
uterine response. At the higher dose, the extent of oestrogenicity
of the extract from the transgenic extract was comparable to E2,
thus raising the possibility that it could have substantial bone
sparing effect under E2 deficiency. The hallmark of postmenopausal osteoporosis is decrease in trabecular bone with accompanying deterioration of microarchitecture (Weinstein and
Hutson, 1987). Indeed, high levels of oestrogenicity translated
to osteoprotection in Ovx mice as assessment of trabecular bone
at femur epiphysis showed that only the higher dose of the
transgenic extract (100 mg/kg) prevented trabecular loss and
microarchitectural destruction and not the lower dose (50 mg/
kg), which had marginal oestrogenicity. Remarkably, the preservation of trabecular bone by transgenic extract at 100 mg/kg
dose was complete (comparable to the sham or E2 supplemented
group), which further suggested that the skeletal effect was
caused by the oestrogenicity of the extract as the later effect was
also equivalent to E2. A complete skeletal preservation of Ovx
mice by transgenic extract (100 mg/kg dose) appeared to be due
to complete suppression of osteoclast number and surface as
these parameters were comparable with the sham and Ovx + E2
groups. Because endometrial hyperplasia or excessive cell growth
in the uterus is an unfavourable condition for the treatment of
postmenopausal osteoporosis, oestrogenicity of the transgenic

extract is a contra-indication for human use as it could increase

the risk of uterine cancer. Nonetheless, positive skeletal effects of
the transgenic extract provide a proof-of-concept towards
effective bone preservation via the enrichment of phyto-oestrogens by pathway engineering in plants.
Cellular mechanism of osteoprotective effect appears to be the
suppression of osteoclast function by transgenic extract evident
from huge decrease in ex vivo osteoclast formation in BMC of
Ovx + T group compared with Ovx + vehicle group. E2 deficiency
triggers osteoclast activation with consequent increase in bone
resorption, and antiresorptive agents such as selective oestrogen
receptor modulators or bisphosphonates constitute initial lines of
therapy for osteoporosis (DAmelio et al., 2008; Pacifici, 1996).
Antioxidants counteract the increased production of osteoclasts
due to ROS action on the precursor cells and establish a causal
link between the oxidative stress on the body and bone loss (Lean
et al., 2003). E2 is known to have antioxidant property and Ovx
mice had lower serum antioxidant levels compared with sham. In
comparison with Ovx + vehicle, all treatment groups had
increased serum antioxidant levels; however, the level was
highest in transgenic extract (100 mg/kg) followed by E2, and
both were higher than the sham. From these data, it appeared
that strong antioxidant effect of transgenic extract coupled with
oestrogenic response contributed to its substantial bone-conserving effect in the Ovx mice. The data also suggest that enhancement in the isoflavonoid content in nonleguminous plants
through AtMYB12 may lead to the development of plants for
better bone health.
In last two decades, different crops have emerged as important
target for value addition with various flavonoids through genetic
engineering. In general, edible portion of these crops is highly
deficient in health-beneficial flavonoids and specially isoflavonoids. Although efforts have been made through using different
approaches including bifunctional enzymes and gene pyramiding,
limited success has been achieved. As AtMYB12 transcription
factor has been used to develop transgenic plants other than
tobacco (Butelli et al., 2008; Luo et al., 2008), with enhanced
level of flavonoids that are substrate for IFS, our approach based
on co-expression of GmIFS1 and AtMYB12 holds great promise in
metabolic engineering of isoflavones as well as flavonols in
different crops of interest.

Experimental procedures
Plasmid construction and plant transformation
The plant expression construct for constitutive expression of
AtMYB12 was developed in pBI121 vector (pBI121-AtMYB12) as
described by Misra et al. (2010b). The full-length GmIFS1 cDNA
was isolated through RT-PCR of total RNA of soybean seedling. For
the purpose, a set of primers (GmIFS1F and GmIFS1R) were
designed using sequence information of GmIFS1 mRNA
(AF195798) with modified nucleotides to accommodate restriction
sites for Xba1 and Sac1 enzymes (Fermentas Life Sciences) at
forward and reverse primers, respectively. The full-length cDNA of
GmIFS1 was cloned in pBI121, resulting into pBI121-GmIFS1 binary
vector. For the development of construct allowing constitutive
co-expression of GmIFS1 and AtMYB12, the plant expression
constructs pBI121-GmIFS and pBI121-AtMYB12 were explored.
Firstly, pBI121-AtMYB12 plasmid was digested with EcoR1 and
HindIII to remove CaMV-AtMYB12-NOS-T cassette. The purified
cassette was end-filed with Klenow enzyme. The plasmid pBI121GmIFS was digested with HindIII enzyme followed by purification.

2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 6980

Isoflavone metabolic engineering and improved bone health 77

The purified digested vector was end-filed with Klenow enzyme
followed by CIAP (Calf intestinal alkaline phosphatase) treatment
and purification. The purified vector was ligated with CaMVAtMYB12-NOS-T cassette. The ligation mixture was transformed in
competent E. coli (DH5a) (Misra et al., 2010b) cells. Plasmids from
recombinant E.coli were isolated and sequenced to verify constructs. Screening of putative transformants, grown in presence of
kanamycin, was performed using colony PCR, primed with CaMV
forward and NosT reverse primers followed by gene-specific
primers. The plasmid was isolated from positive colonies and
subjected to further confirmation through restriction digestion as
well as through sequencing.
These plant expression constructs in pBI121 were transformed
into Agrobacterium LBA4404 strain through electroporation.
Nicotiana tabacum cv. Petit Havana was transformed using
agrobacterium harbouring desired constructs (pBI121-GmIFS1
and co-expression construct,) using protocol as described by
Horsch et al. (1985). Several transgenic lines were regenerated in
case of co-expressing construct, and these were studied for
transgenic nature and petal pigmentation in T0 generation. Based
on reduction in petal pigmentation and high-level expression of
transgenes, three homozygous transgenic lines (1, 2 and 3) were
selected for further study.

Gene expression analysis

Total RNA was extracted from young leaves and flower petals of
flowering tobacco plants using the Spectrum Plant Total RNA kit
(Sigma-Aldrich), which was subsequently treated with RNase-free
DNase (Fermentas Life Sciences). Total RNA was subjected to
reverse transcription reaction to generate first-strand cDNA using
oligo (dT) primers (MBI Fermentas). RT-PCR analysis of a set of
selected genes was carried out using 2X PCR Master mix
(Fermentas Life Sciences). The lists of selected genes and
oligonucleotide primers used in the study are provided in Table
S1. The primers for the tobacco ubiquitin gene were used as a
loading control to ensure that equal amounts of cDNA were used
in all the reactions. PCR was carried out using the following cycle
conditions: an initial denaturation at 94 C for 2 min, 30 cycles at
94 C for 30 s, 55 C for 30 s and 72 C for 30 s, followed by a
final 5-min extension at 72 C.
For real-time expression analysis, PCR mix contained 1 lL of
diluted cDNA (10 ng), 10 lL of 2X SYBR Green PCR Master Mix
(Applied Biosystems, CA) and 200 nM of each gene-specific primer
in a final volume of 20 lL. PCRs with no template controls were
also performed for each primer pair. Expression of different genes
involved in flavonoid biosynthesis was studied through Applied
Biosystems 7500 Fast Real-time PCR System. All the PCRs were
performed under following conditions: 20 sec at 95 C, 3 sec at
95 C and 40 cycles of 30 sec at 60 C in 96-well optical reaction
plates (Applied Biosystems). The specificity of amplicons was
verified by melting curve analysis (6095 C) after 40 cycles. Three
technical replicates were analysed for each cDNA.
SYBR green chemistry was used for quantitative determination
of a pair of osteoclast-specific genes following an optimized
protocol described before (Siddiqui et al., 2010; Swarnkar et al.,
2011). The design of sense and antisense oligonucleotide primers
was based on published cDNA sequences using the Universal
ProbeLibrary (Roche Applied Sciences). Primer sequences are
listed in Table S1. cDNA was synthesized with the RevertAid
cDNA synthesis kit (Fermentas, Austin, TX) using 2 lg total RNA
in 20-lL reaction volume. For qPCR, the cDNA was amplified

using Light Cycler 480 (Roche Molecular Biochemicals, Indianapolis, IA).

HPLC analysis for quantification for flavonoids and

isoflavonids
Analysis of the flavonols was carried out as described earlier
(Niranjan et al., 2011) with modifications (Pandey et al., 2012b).
Flavonols and isoflavones in the plants were determined either as
aglycones or as their glycosides by preparing acid-hydrolysed or
nonhydrolysed extracts, respectively. For preparation of acidhydrolysed extract, plant material was extracted with 80%
ethanol overnight at room temperature with brief agitation. The
filtrates were evaporated to 1.0 mL, and three volume of HCl
(1 M) was added followed by incubation at 94 C for 2 h to
hydrolyse any conjugate forms of flavonoids. After hydrolysis,
samples were extracted with ethyl acetate, evaporated to dryness
and resuspended in 80% methanol. Separation for qualitative
and quantitative analysis of isoflavones and other flavonoids in
hydrolysed methanolic extracts of plant tissue was performed by
HPLC-PDA with a Shimadzu (Kyoto, Japan) LC-10 system comprising an LC-10 AT dual pump system, an SPD-M20A PDA
detector (254 nm) and rheodyne injection valve furnished with a
20-lL sample loop. Compounds were separated on a
4.6 9 250 mm, i.d. 5-lm pore size Marck column protected
by guard column containing the same packing. The mobile phase
was a gradient prepared from 0.5% (v/v) phosphoric acid in
HPLC-grade water (Component A) and methanol (component B).
Before use, the compounds were filtered through 0.45-lm nylon
filters and re-aerated in an ultrasonic bath. Data were integrated
by Shimadzu class VP series software, and results were obtained
by comparison with standards. Results are mean values from
three replicate analysis of the same sample.

Mass spectrometry analysis

Mass spectrometry analysis was carried out as described by
Pandey et al. (2012b). Analysis was performed with an Applied
Biosystems (Ontario, Canada), for ionization API 2000 triple
quadrupole mass spectrometer. A turbo ion-spray source was
used in negative-ion mode with the settings: nebulizer gas (N2),
15 arbitrary units; curtain gas (N2), 11 arbitrary units; collision gas
(N2), 12 arbitrary units; focusing potential, _450 V; entrance
potential, _11 V; declustering potential, 2080 V; and collision
energy, 1550 arbitrary units. Full scan acquisition was performed
between m = z 100 and 650U with a cycle time of 3 s. Product
ion scans of molecule was conducted to confirm compound
structure. The product ions were produced by collision-associated
dissociation of selected precursor ions in a collision cell. Experiments were performed with the quadrupole (Q1) operated at
unit resolution.

Assessment of skeletal effect in mice

Leaves from the AtMYB12- and GmIFS1-co-expressing tobacco
transgenic line and WT tobacco plants were air-dried and ground
to fine powder. The leaf powder was extracted overnight with 10
volumes of absolute ethanol. The extract was filtered, and plant
debris was again extracted with same volume of absolute alcohol.
The pooled alcoholic extracts were evaporated to dryness in a
rotavapour. This ethanolic extract was used for in vivo testing of
osteogenic activity in rats. All fine chemicals including 17-b
estradiol and primers were purchased from Sigma-Aldrich
(St.Louis, MO).

2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 6980

78 Ashutosh Pandey et al.

All animal procedures were approved by Institutional Animal
Ethical Committee at Central Drug Research Institute. Adult Balb/
c mice (910 weeks old) were taken for the study. All mice were
housed at 25 C, in 12-h light/12-h dark cycles, maintained under
specific pathogen-free conditions and fed sterilized food, that is,
standard rodent chow diet (Khan et al., 2012; Swarnkar et al.,
2011) and autoclaved water ad libitum. Eight mice per group
were taken for the study. The groups were sham-operated (ovary
intact) mice, given vehicle (gum acacia in distilled water);
ovariectomy + vehicle; ovariectomy + 50 mg/kg and ovariectomy + 100 mg/kg body weight of both wild and transgenic
plant extracts and ovariectomy + 17b estradiol (10 lg/kg). Mice
were treated once daily for 4 weeks by oral gavage. Initial and
final body and uterine weights were recorded. For the uterotropic
assay and bilateral ovariectomy, we followed our previously
published protocol (Swarnkar et al., 2011).

Microcomputed tomography (lCT) analysis and

determination of serum antioxidant levels
lCT scanning and analysis of excised bones were carried out
using the SkyScan 1076 lCT scanner (Aartselaar, Belgium) as
reported previously (Khedgikar et al., 2013; Sharan et al., 2011;
Khan et al., 2012). Serum antioxidant level was measured
according to the manufacturers protocol using antioxidant
measurement kit as reported earlier (Srivastava et al., 2013a).

Ex vivo osteoclastogenesis and qPCR

Bone marrow cells from the various experimental groups was
harvested from the long bones and cultured overnight in a-MEM
supplemented with 10% heat-inactivated foetal bovine serum
(FBS) and 10 ng/mL M-CSF. The nonadherent BMC were
collected, and after centrifugation, these were cultured on 48well tissue culture plates in aMEM plus FBS containing 30 ng/mL
M-CSF and 50 ng/mL RANKL as differentiation inducers. Cultures were maintained for 6 days as reported previously (Khan
et al., 2012). qPCR was performed for assessing the expression
of TRAPC5b and cathepsin-K from the total RNA of differentiated osteoclasts. Primers were designed using the Universal
ProbeLibrary (Roche Applied sciences) for genes viz., TRAPC5b,
cathepsin-K and GAPDH (internal control) (Refer to Table S1 for
oligonucleotides sequence). cDNA was synthesized with a
RevertAid cDNA synthesis kit (Fermentas, Austin) using 2 lg
total RNA. SYBR green chemistry was used to perform quantitative determination of relative expression of transcripts for all
genes. All genes were analysed using the Light Cycler 480
(Roche Molecular Biochemicals, Indianapolis, IN) real-time PCR
machine.

Determination of osteoclast number and surface

Femurs were decalcified and 5-lm longitudinal sections through
epiphysis region were stained for TRAP (Wei et al., 2013).
Histomorphometric analysis was performed using BIOQUANT
OSTEO MPR software version 12.5.6 (Nashville TN). The number
of osteoclasts (OC)/bone perimeter (N.Oc/B.Pm) and OC surface/
bone surface (%) were analysed.

Statistical analysis
Data are expressed as mean  SEM unless otherwise indicated.
The data obtained in experiments with multiple treatments were
subjected to one-way ANOVA followed by post hoc Newman
Keuls multiple comparison test of significance using GraphPad
Prism 3.02 software (La Jolla, CA). Qualitative observations have

been represented following assessments made by three individuals blinded to the experimental designs.

Acknowledgement
Research was supported by Council of Scientific and Industrial
Research, New Delhi, in the form of Network projects PlaGen
(BSC-0107) and Center for Research in Anabolic Skeletal Targets
in Health and Illness (ASTHI, BSC-0201). AP acknowledges
Council of Scientific and Industrial Research, New Delhi, for
Senior Research Fellowship.

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Supporting information
Additional Supporting information may be found in the online
version of this article:
Figure S1 Phytochemical analysis of the methanolic extracts of
leaves of different transgenic lines. Compounds were quantified
by separating methanolic leaf extracts of WT and transgenic lines
using HPLC. The graph shows values  SD of three leaves from
each of the independent transgenic line.
Figure S2 HPLC profiles illustrating the accumulation of genistein
and other flavonoids in transgenic and WT tobacco acid-hydrolysed extracts. (A) Standards: rutin (1), quercetin (2), genistein (3)
and kaempferol (4). (B) WT tobacco leaf. (C) GmIFS1-expressing
tobacco leaf. (D) AtMYB12-expressing tobacco leaf. (E) AtMYB12- and GmIFS1-co-expressing tobacco leaf.
Figure S3 Negative-ion ESI-MS spectrum of standard genistein
(A) and genistein of leaf extract (B) from transgenic plant coexpressing AtMYB12 and GmIFS1.
Figure S4 Leaf extract from transgenic plant has oestrogenicity.
Transverse sections of uterus (5 lm) were stained with H&E and
representative images (40 9 magnifications) of different experimental groups are shown. Quantification of data is provided in
Table 2.
Table S1 List of oligonucleotides and their use in the study.

2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 6980