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Co-expression of Arabidopsis transcription factor,AtMYB12, and soybean isoflavone synthase, GmIFS1,genes in tobacco leads to enhanced biosynthesis ofisoflavones and flavonols resulting in osteoprotectiveactivityAshutosh Pandey1,, Prashant Misra1,,, Mohd P. Khan2, Gaurav Swarnkar2, Mahesh C. Tewari2, SwetaBhambhani1, Ritu Trivedi2, Naibedya Chattopadhyay2 and Prabodh K. Trivedi1,*

1Council of Scientific and Industrial Research-National Botanical Research Institute (CSIR-NBRI), Lucknow, India2CSIR-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 [email protected],

[email protected])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.

SummaryIsoflavones, 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 AtMYB12and GmIFS1

(soybeanIFS) 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

andGmIFS1 but not wild-type extract exhibited significant conservation of trabecularmicroarchitecture, 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 ubiqui-tous 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 (Ververidiset 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 Vanderley-

den, 1995; Pueppke, 1996, Dixon, 1999; Subramanian et al.,

2007). Biosynthesis of these molecules is known to be induced bydefence 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, osteoporo-

sis and menopausal symptoms (Cornwell et al., 2004; Dixon,

2004; Srivastavaet al., 2013a,b; Tyagiet al., 2010; Pandeyet 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-osteo-

porotic and antiproliferative activities in human and animal

systems (Birtet al., 2001; Iwasaki et al., 2008; Miadokova et al.,

2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd 69

Plant Biotechnology Journal (2014)12, pp. 6980 doi: 10.1111/pbi.12118


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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 sug-

gested 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 ofisoflavones (Miadokova, 2009). Owing to such health-beneficial

properties of isoflavones, it is desirable to incorporate or

improve their biosynthesis in commonly consumed nonlegumi-

nous 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 agly-

cone isoflavones are often conjugated with glycosyl and malonyl

residues by glycosyl- and malonyltransferases enzymes (Dhaubh-

adel 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 (Akashiet 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 nonlegu-

minous plants such asArabidopsis, tobacco,Petunia, lettuce and

tomato (Junget al., 2000; Liuet al., 2002; Liuet 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 leaftissue. 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 (Yiet al., 2010). However, overexpression of GmMYB176in

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-regu-

lated 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 utilizedAtMYB12transcription 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 miceconfers 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 differentgenes

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.

(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 carryingAtMYB12andGmIFS1genes in pBI121 vector used fortobacco transformation.

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

Ashutosh Pandeyet al.70


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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. TheAgrobacterium-mediated tobacco trans-

formation was carried out, and several independent kanamycin-

resistant 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 aftertransformation with the constructs havingAtMYB12and GmIFS1

alone showed the presence of respective genes only. RT-PCR

analysis of selected transgeniclines suggestedhigh-level expression

ofAtMYB12andGmIFS1in the co-expressing lines (Figure 2c).

Modulated expression of genes involved in flavonoidbiosynthesis 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 phenylpropa-

noid pathway and flavonoid pathway such asPAL,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, enhance-

ment in the expression of the genes was much more in leaf in

comparison with petals. These results establish that the expres-

sion of AtMYB12 in AtMYB12 and GmIFS1 co-expressing or

AtMYB12alone 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 unhydro-

lysed methanolic extracts suggested rutin as the most abundantflavonol 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 acid-

hydrolysed 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)

(c)

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 semi-

quantitative RT-PCR using total RNA from leaves

of different transgenic lines.


Isoflavone metabolic engineering and improved bone health 71


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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-alone-

or 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 differenttransgenic lines. Petals of GmIFS1-expressing and AtMYB12-

and 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, serumantioxidant level and trabecular microarchitecture inOvx 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 (desig-

nated 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 measuredtotal 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 numer-

ous 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

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.




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

(Tb.Th) and connectivity density (Conn.D) and increased trabec-

ular 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 osteoclastsfrom 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 nuclearfactor kappa-B ligand) and M-CSF (macrophage colony-stimulat-

ing factor) and examined the expression of major osteoclasts

differentiation markers including tartrate-resistant acid phospha-

tase 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 defi-

ciency. 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-

pressedex vivoosteoclastogenesis 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 perim-

eter (N.Oc/B.Pm) and OC surface/bone surface (%) was signifi-

cantly 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 transgenic-

extract-treated Ovx rats (Ovx + T50) compared with Ovx + vehi-

cle. 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 photo-

micrograph 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

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.




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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; Misraet al., 2010a;

Shihet 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 indifferent plant parts with its enhanced activity in petals as evident

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

Conversely, leaves have lesser flavonoid pathway activity, and

therefore, the expression of legume IFSgenes 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 expres-

sion pattern of these transcription factors has been attributed to

the differential regulation of genes involved in flavonoid biosyn-thesis under developmental and environmental cues. It has been

established that AtMYB12 modulates genomewide expression

and metabolome in transgenic tobacco lines regulate phenyl-

propanoid 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-expressingAtMYB12 and GmIFS1 genes and thereby activat-

ing 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 synthesizedin transgenic lines developed, in this study, is the highest amount

(a)

(c)

(d)

(b)

Figure 5 Body weight, serum antioxidant levels andlCT of femur epiphysis of mice. (a) Body weight gain (%) at the end point, (b) total serum antioxidant

levels at the end point, (c) representativelCT images of femur epiphysis at the end point and (d) quantification oflCT 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.




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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 legumeIFS

genes (Figure 4). Recently, Ogo et al. (2013) used constructs

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 IFSfor 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 glycocon-

jugates accumulate at substantial amounts in leaves of

co-expressing transgenic lines, but their amounts are not com-

parable 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

Figure 6 Transgenic extract diminishesex vivo osteoclastogenesis inmice. 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.

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

OC Surface/bone

surface (%)

3.7 0.21a 7.8 1.3x 8.0 1 .6x 8.2 0.68x 6.3 0.75z 3.8 0.64a 3.6 1.6a

cP < 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

Parameter Sham Ovx + Veh Ovx + WT50 Ovx +WT10 0 Ovx + T50 Ovx + T100 Ovx + E2

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 87.0 10.13y,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 100.3 3.63x,a

Luminal area (lm2) 12.5 0.622a 7.3 0.32x 8.2 0.71x 9.6 0.70x,a 9.09 0.24a 12.2 1.09a 9.7 0.69x,a

Luminal epithelium height (lm) 0.40 0.007a 0.11 0.010x 0.11 0.003x 0.12 0.002x 0.20 0.003x,a 0.34 0.038x,a 0.26 0.053x,a

cP < 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.




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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 dehydra-

tase) 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

ofF3H 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

GmIFS1in 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 oftrabecular bones (Boydet 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 legumi-

nous plants (Justesenet 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 postmeno-

pausal osteoporosis is decrease in trabecular bone with accom-

panying deterioration of microarchitecture (Weinstein and

Hutson, 1987). Indeed, high levels of oestrogenicity translated

to osteoprotection in Ovx mice as assessment of trabecular boneat 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 preser-

vation 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-oestro-

gens 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 (DAmelioet 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 washighest 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-conserv-

ing effect in the Ovx mice. The data also suggest that enhance-

ment 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 isoflavo-

noids. 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 ofGmIFS1andAtMYB12holds 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

AtMYB12was developed in pBI121 vector (pBI121-AtMYB12) asdescribed by Misraet 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 wascloned in pBI121, resultinginto 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 pBI121-

GmIFS was digested withHindIII enzyme followed by purification.




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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 CaMV-

AtMYB12-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 con-

structs. 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

Horschet 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. Basedon 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 wereperformed 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; Swarnkaret 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, Indianap-

olis, IA).

HPLC analysis for quantification for flavonoids andisoflavonids

Analysis of the flavonols was carried out as described earlier

(Niranjanet 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 acid-

hydrolysed 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 com-prising 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. Exper-iments 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).




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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 ovariec-

tomy + 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 anddetermination of serum antioxidant levels

lCT scanning and analysis of excised bones were carried outusing the SkyScan 1076 lCT scanner (Aartselaar, Belgium) as

reported previously (Khedgikar et al., 2013; Sharanet al., 2011;

Khan et al., 2012). Serum antioxidant level was measured

according to the manufacturers protocol using antioxidant

measurement kit as reported earlier (Srivastavaet 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 48-

well tissue culture plates in aMEM plus FBS containing 30 ng/mL

M-CSF and 50 ng/mL RANKL as differentiation inducers. Cul-

tures 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 differenti-

ated 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 quanti-

tative 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 individ-

uals 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 S2HPLC profiles illustrating the accumulation of genistein

and other flavonoids in transgenic and WT tobacco acid-hydro-

lysed 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) At-

MYB12- 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 co-

expressingAtMYB12 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 exper-

imental groups are shown. Quantification of data is provided in

Table 2.

Table S1 List of oligonucleotides and their use in the study.



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