Research

PAP1 transcription factor enhances production of

phenylpropanoid and terpenoid scent compounds in rose flowers
Michal Moyal Ben Zvi1, Elena Shklarman1, Tania Masci1, Haim Kalev2, Thomas Debener3, Sharoni Shafir2,
Marianna Ovadis1 and Alexander Vainstein1
1
Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel;
2
B. Triwaks Bee Research Center, Department of Entomology, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel;
3
Institute for Plant Genetics, Molecular Plant Breeding, Leibniz University of Hannover, Hannover D-30419, Germany

Summary
Author for correspondence: • Floral scent is a complex trait of biological and applied significance. To evaluate whether
Alexander Vainstein scent production originating from diverse metabolic pathways (e.g. phenylpropanoids and
Tel: +972 8 9489082 isoprenoids) can be affected by transcriptional regulators, Arabidopsis PRODUCTION OF
Email: vain@agri.huji.ac.il
ANTHOCYANIN PIGMENT1 (PAP1) transcription factor was introduced into Rosa hybrida.
Received: 31 January 2012 • Color and scent profiles of PAP1-transgenic and control (b-glucuronidase-expressing) rose
Accepted: 24 March 2012 flowers and the expression of key genes involved in the production of secondary metabolites
were analyzed. To evaluate the significance of the scent modification, olfactory trials were
New Phytologist (2012) 195: 335–345 conducted with both humans and honeybees.
doi: 10.1111/j.1469-8137.2012.04161.x • In addition to increased levels of phenylpropanoid-derived color and scent compounds
when compared with control flowers, PAP1-transgenic rose lines also emitted up to 6.5
times higher levels of terpenoid scent compounds. Olfactory assay revealed that bees and
Key words: olfactory, phenylpropanoid,
PRODUCTION OF ANTHOCYANIN
humans could discriminate between the floral scents of PAP1-transgenic and control
PIGMENT1 (PAP1), Rosa hybrida, scent, flowers.
terpenoid. • The increase in volatile production in PAP1 transgenes was not caused solely by transcrip-
tional activation of their respective biosynthetic genes, but probably also resulted from
enhanced metabolic flux in both the phenylpropanoid and isoprenoid pathways. The mecha-
nism(s) governing the interactions in these metabolic pathways that are responsible for the
production of specialized metabolites remains to be elucidated.

include basic helix–loop–helix (bHLH) transcription factors

Introduction
Roses are among the world’s most important ornamentals,
including 200 species and > 18 000 cultivars used as cut flowers,
garden and pot plants (Gudin, 2000). Modern rose varieties
possess varied color and scent profiles deriving from their diverse
progenitors (Schulz, 2003; Verhoeven & Brandenburg, 2003).
Flavonoids, carotenoids or both determine the color of rose
flowers, which is dependent on the variety: yellow and orange
hues are commonly contributed by carotenoids; other shades of
red, pink, violet and mauve are generally contributed by flavo-
noids (Schulz, 2003). The main flavonoids contributing to rose
petal color are the anthocyanins cyanidin 3,5-diglucoside, pelarg-
onidin 3,5-glucoside and paeonidin 3,5-diglucoside (Ogata
et al., 2005). Co-pigments, such as flavonol glycosides, also
greatly influence the flower’s final color (Jay et al., 2003). The
molecular basis for the transcriptional regulation of flavonoid
biosynthesis is well established, and has been shown to operate
through complexes of transcription factors. These complexes
which interact with R2R3 MYB proteins, and WD40 proteins
required for the activity of the regulatory complex (Koes et al.,
2005; Hichri et al., 2011). PRODUCTION OF ANTHO-
CYANIN PIGMENT1 (PAP1) is a Myb transcription factor from
Arabidopsis thaliana which has been shown previously to exert
broad activation of the phenylpropanoid pathway (Borevitz
et al., 2000; Mathews et al., 2003; Matousek et al., 2006; Ben
Zvi et al., 2008a; Li et al., 2010).
Analyses of floral scent in numerous rose varieties have led to
the identification of over 400 volatile compounds that derive
from terpenoid, phenylpropanoid ⁄ benzenoid and fatty acid path-
ways (Guterman et al., 2002; Scalliet et al., 2008). On the basis
of their chemical structure, these volatile compounds can be
classified into five groups: hydrocarbons, alcohols, esters,
aromatic ethers and others (including aldehydes, aliphatic chains,
rose oxides and norisoprenes) (Verhoeven & Brandenburg,
2003). In nature, the unique combination of scent molecules
emitted by flowers is detected by the olfactory receptors of

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336 Research
insects, enabling them to find and visit their flower(s) of choice
(Shalit et al., 2004; Smith et al., 2006). Moreover, the diurnal
pattern of floral volatile emission from wild species of the genus
Rosa is synchronized with the active hours of its bee pollinator
(Hendel-Rahmanim et al., 2007).
The characterization of the pattern of scent production in rose
has revealed the existence of numerous levels of complexity in the
regulation of floral volatile production (Hendel-Rahmanim
et al., 2007). Attempts to characterize rose’s genetic machinery
of scent production have led to the identification and character-
ization of several genes coding for enzymes responsible for the
production of floral scent compounds, among them GER
MACRENE D SYNTHASE (GDS), CAROTENOID CLEAVAGE
DIOXYGENASE1 (CCD1), ORCINOL O-METHYLTRANS
FERASE1 (OOMT1), GERANIOL ⁄ CITRONELLOL ACETYL
TRANSFERASE1 (AAT1) and PHENYLACETALDEHYDE
SYNTHASE (PAAS), which catalyze the production of germac-
rene D, b-ionone, orcinol methyl ether, geranyl acetate and
phenyl acetaldehyde, respectively (Guterman et al., 2002; Lavid
et al., 2002; Shalit et al., 2003; Farhi et al., 2009; Huang et al.,
2009). In addition to structural scent-related genes, several Myb
transcription factors putatively involved in the regulation of floral
scent production have been identified in roses (Guterman et al.,
2002; Yan et al., 2010).
High commercial value is a major driving force for the
breeding of modern rose cultivars (Chandler & Lu, 2005).
Genetic engineering approaches have been implemented in rose
for the alteration of traits towards, for example, resistance to
pathogens, improved morphological traits and color modification
(Chandler & Lu, 2005; Tanaka et al., 2005; Chandler &
Tanaka, 2007; Katsumoto et al., 2007). Although fragrance is
still among the traits most identified with rose species, it is almost
absent in many modern cut flower varieties (Chandler & Lu,
2005; Chandler & Tanaka, 2007). Only c. 20% of all rose species
known today are classified as ‘fragrant’ and 30% are not fragrant
at all (Schulz, 2003). Recently, progress has been made towards
establishing a marker-assisted breeding platform for scented rose
varieties, and several loci associated with specific scent patterns in
roses have been mapped (Spiller et al., 2010).
Rosa hybrida ‘Pariser Charme’ has dark red flowers as a result
of anthocyanin accumulation (Yokoi, 1974), which produce
several volatile compounds deriving from diverse biosynthetic
origins (Schulz, 2003). The availability of an embryogenic
callus-based genetic transformation system (Dohm et al., 2001)
developed for this cultivar enabled us to introduce PAP1 into
‘Pariser Charme’ rose plants to investigate the extent of the effect
of PAP1 on the production of floral volatiles from diverse
biochemical origins. Remarkably, the expression of PAP1 in flow-
ers of ‘Pariser Charme’ roses led to increased levels of volatile
compounds originating from various biosynthetic origins, for
example, phenylpropanoids, monoterpenes, sesquiterpenes and
norisoprenes; this increase was not solely dependent on the
transcriptional activation of the respective structural genes. The
change in the volatile profile of PAP1-transgenic rose was olfacto-
rily significant, and distinguishable by both humans and
honeybees.
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Materials and Methods
Construction of chimeric genes, plant transformation and
regeneration
PAP1 (GenBank accession no. AF325123) coding for the Myb
transcription factor was cloned from Arabidopsis ‘Colombia’
DNA and inserted into the binary vector pCGN1559, as
described in Ben Zvi et al. (2008a). Agrobacterium tumefaciens
strain AGLO (Lazo et al., 1991), carrying the binary plasmid
pCGN1559 or pCGN7001 (containing the uidA gene coding
for b-glucuronidase (GUS) (Comai et al., 1990)), was used for
the stable transformation of rose to generate PAP1-transgenic or
control, GUS-transgenic plants, respectively. Transformation of
embryogenic callus initiated from Rosa hybrida L. cv Pariser
Charme (Dohm et al., 2001), selection and regeneration of trans-
genic plants were conducted as described by Dohm et al. (2001).
Callus was cultured in Murashige and Skoog basal medium
(Sigma) supplemented with 0.01 mg l)1 indole-3-butyric acid
(IBA), 2 mg l)1 6-benzylaminopurine (BAP) and 0.1 mg l)1
gibberellic acid (GA3) for shoot induction, and 0.01 mg l)1 IBA,
0.5 mg l)1 BAP and 0.2 mg l)1 GA3 for shoot multiplication
and elongation. Shoot rooting was induced by subculture on
half-strength Murashige and Skoog medium supplemented with
0.1 mg l)1 IBA. The regenerated plantlets were planted into a 1 :
1 mixture of standard potting soil and perlite for glasshouse adap-
tation. Plants were then transferred to 18-cm-diameter containers
with potting soil (Shaham, Giva’t Ada, Israel).
Plant growth and plant material
Plants were grown in a glasshouse under 25C : 22C day :
night temperatures and a natural photoperiod for 2 yr in
Rehovot, Israel. Control GUS-expressing plants were intermixed
with the PAP1-transgenic lines. Flowers were analyzed at devel-
opmental stage 4 (when the sepals are beginning to retract, but
the petal whorl is still closed) and stage 7 (the outer petal whorl is
open, but the inner petal whorl is still closed and the reproductive
organs are not yet visible).
Histochemical assay of GUS expression
GUS activity was analyzed histochemically as described previ-
ously (Ben Zvi et al., 2008b). Tissue samples were incubated for
a few hours to overnight at 37C in a 0.1% (w ⁄ v) X-Gluc
(5-bromo-4-chloro-3-indolyl b-D-glucuronic acid sodium salt;
Biosynth Inc., Staad, Switzerland) solution containing 0.1 M
sodium phosphate buffer (pH 7.0), 10 mM EDTA and 0.1%
(w ⁄ v) Triton X-100. When necessary, green tissues were
bleached, after staining, by immersion in 50% ethanol for a few
hours, followed by several washes with 70% ethanol.
Anthocyanin content
To determine the anthocyanin content, three tissue replicates
(each 100 mg fresh weight (FW)) from leaves and stage 4 flowers
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of control and PAP1-transgenic lines 6, 11, 12 and 13 were
extracted in 1 ml of methanol containing 1% (v ⁄ v) HCl. Follow-
ing overnight incubation in the dark at ) 4C with shaking at
150 rpm, the extract was centrifuged at 10 500 g for 10 min.
The anthocyanin content in the supernatant was determined on
the basis of the absorbance at 530 nm (Ben Zvi et al., 2008a).
RNA extraction and real-time quantitative PCR analysis
Gene expression analyses were conducted on PAP1-transgenic lines
6, 11, 12 and 13 and GUS-transgenic control plants. Petals at stage
4 were collected at 21:00 h, when the genes involved in scent pro-
duction are optimally expressed (Guterman et al., 2002). Flower
and leaf tissues (100 mg) were extracted as described previously
(Guterman et al., 2002), and total RNA was treated with RNase-free
DNase (Fermentas, Glen Burnie, MD, USA). First-strand cDNA
was synthesized using 1 lg of total RNA, oligo d(T) primer and
Reverse Transcriptase ImProm-II (Promega). Control samples
were generated without the addition of reverse transcriptase to
the reaction. PCR was performed for 40 cycles (94C for 15 min
and then cycling at 94C for 10 s, 60C for 30 s and 72C for 20
s). To confirm that the analyzed samples were not contaminated
with DNA, real-time PCR using ACTIN primers was also
conducted with samples generated without reverse transcriptase.
The primers (forward and reverse, respectively) used for
PHENYLALANINE AMMONIA LYASE (PAL; GenBank
accession no. BQ105227) were 5¢-GCTAGGGCTGCATACG-
AGAG-3¢ and 5¢ GTTCCAACCACTGAGGCAAT-3¢; for
CHALCONE SYNTHASE (CHS AB038246), 5¢-GCGACAC-
CCATCTCGATAGT-3¢ and 5¢-TCGTTGAGGCTCTTCTC-
GAT-3¢; for CHALCONE ISOMERASE (CHI; AY040321),
5¢-GCCAGCAATACTCGGAGAAG-3¢ and 5¢-AAGCCAATC-
GTCAATGATCC-3¢; for FLAVANONE 3-HYDROXYLASE
(F3H; BQ105585), 5¢-GCAACGAAATTCCGATCATT-3¢ and
5¢-GAACTCTCTGGCGAGACTGG-3¢; for DIHYDROFLAV-
ONOL-4-REDUCTASE (DFR; D85102), 5¢-GCAACGAAAT-
TCCGATCATT-3¢ and 5¢-GAACTCTCTGGCGAGACTG-
G-3¢; for ANTHOCYANIN SYNTHASE (ANS; BI977949),
5¢-GCTCGTCAACAAGGAGAAGG-3¢ and 5¢-GGTAGAGG-
CGAGAGCTTCCT-3¢; for GERMACRENE D SYNTHASE
(GDS; BQ105086), 5¢-CTCCGCCAGTTCAAACAAGT-3¢
and 5¢-TTGTAACCATGCTGCCTCAG-3¢; for CAROTEN-
OID CLEAVAGE DIOXYGENASE1 (CCD1; EU327776),
5¢-CGAAAATTGAGGTTGGAGGA-3¢ and 5¢-GCATGGAA-
CCCATATGGAAC-3¢; for EUGENOL SYNTHASE (EGS),
5¢-CTTTAATGGCGGCGATGAT-3¢ and 5¢-AACCCGGCC-
AAGTCTAAAGT-3¢; for PHENYLACETALDEHYDE SYNTH-
ASE (PAAS; DQ192639), 5¢-CCAGAATTTCGGCATTT-
CAT-3¢ and 5¢-TCAAAAACTCCGGATTCGTC-3¢; for GER-
ANIOL ⁄ CITRONELLOL ACETYL TRANSFERASE1 (AAT1;
BQ106456), 5¢-CCCACAACGTATTTCCCAGT-3¢ and 5¢-AT
GCCTGCTTCGAAATCATC-3¢; for ORCINOL O-METH-
YLTRANSFERASE1 (OOMT1; AF502433), 5¢-CAATCCATC-
CAACCAAATCC-3¢ and 5¢ ATCGTTTTGGAACCAAGTG-
C-3¢; for ACTIN (AB239789), 5¢-CAATGTGCCCGCTATG-
TATG-3¢ and 5¢-CTGTAAGGTCACGTCCAGCA-3¢. cDNA
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amounts were standardized to the expression of two reference
genes: ACTIN (AB239789) and a-TUBULIN (AF394915); as
these gave similar results, only those standardized to ACTIN are
shown. Real-time quantitative PCR was performed in the
presence of SYBR Green I dye (ABgene, Rockford, IL, USA) in a
Corbett Research Rotor-Gene 6000 cycler, and data analysis was
performed using Rotor-Gene 6000 series software 1.7 (QIAGEN
Inc., Valencia, CA, USA).
Collection and extraction of volatile compounds
Volatiles were collected from stage 7 flowers (Guterman et al.,
2006; Hendel-Rahmanim et al., 2007). For headspace analyses,
individual detached rose flowers were enclosed in a 500-ml glass
container with appropriate openings. Emitted volatiles were
collected for 24 h using an adsorbent trap consisting of a glass
tube containing 100 mg Porapak Type Q polymer (80 ⁄ 100
mesh; Alltech, Fresno, CA, USA) and 100 mg 20 ⁄ 40 mesh acti-
vated charcoal (Supelco, Bellefonte, PA, USA), held in place with
plugs of silanized glass wool (Guterman et al., 2006). Trapped
volatiles were eluted using 1.5 ml of hexane, and 2 lg of isobu-
tylbenzene was added to each sample as an internal standard. To
determine the pool sizes of volatile compounds in the petals,
flowers collected at 21:00 h were weighed, ground in liquid
nitrogen and extracted in 10 ml of hexane containing 2 lg of
isobutylbenzene as the internal standard. Following overnight
incubation with shaking at 150 rpm, the extract was centrifuged
at 10 500 g for 10 min and the supernatant was filtered through
a 25-ml syringe with a 0.2-lm sterile nylon filter. Samples were
evaporated using liquid nitrogen to a final volume of 200 ll
before injection into a gas chromatography-mass spectrometry
(GC-MS) instrument.
GC-MS analyses of volatile compounds
GC-MS analyses (1 ll sample) of the extracted volatile com-
pounds were performed in an instrument that included a Pal
autosampler (CTC Analytic, Zwingen, Switzerland), a TRACE
GC 2000 equipped with an Rtx-5SIL MS (Restek; inside
diameter, 0.25 lm; 30 m · 0.25 mm; Bellefonte, PA, USA)
fused-silica capillary column and a TRACE DSQ quadrupole
mass spectrometer (ThermoFinnigan; Hemel Hempstead,
Hertfordshire, UK). Helium was used as the carrier gas at a flow
rate of 0.9 ml min)1. The injection temperature was set to
250C (splitless mode), the interface to 280C and the ion source
adjusted to 250C. The analysis was performed under the following
temperature program: 2 min of isothermal heating at 40C, fol-
lowed by a 5C min)1 oven temperature ramp to 250C and a
final increase from 250 to 270C at 80C min)1. The transfer
line temperature was 250C. The system was equilibrated for 1
min at 70C before injection of the next sample. Mass spectra
were recorded at three scans per second, with a scanning range of
40–450 mass-to-charge ratio and an electron energy of 70 eV.
Compounds were tentatively identified (> 95% match) by com-
parison with the National Institute of Standards and Technology ⁄
Environmental Protection Agency ⁄ National Institutes of Health
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(NIST ⁄ EPA ⁄ NIH) Mass Spectral Library (Data Version: NIST
05, Software Version 2.0 d) using the XCALIBUR v1.3 program
(ThermoFinnigan) library. Further identification of the com-
pounds was based on a comparison of mass spectra and retention
times with those of authentic standards (Sigma) analyzed under
similar conditions.
Scent trials
Two-alternative forced choice (2AFC) scent (orthonasal route)
trials were conducted by untrained panelists differing in sex and
age. Each floral sample contained two open flowers at stage 7.
Each trial was conducted on three different occasions in a room
with a constant temperature of 24 ± 2C and panelists were
blindfolded during the trials. Panelists (n = 24 per trial) were first
presented with either a PAP1-transgenic or control transgenic
floral sample, and then with the reciprocal sample. Panelists were
then asked to indicate the sample with the stronger scent for scent
strength trials, or the sample with the more appealing scent for
the preference trials. A chi-squared test was conducted to examine
the effect of sample presentation order on the tests. A binomial
distribution was conducted on the proportion of expected
responses according to the null hypothesis of the PAP1-transgenic
floral sample being more strongly scented ⁄ preferred over the
control sample. P < 0.05 was considered to be significant.
Proboscis extension response (PER) of honeybees to rose
flower odors
The response of honeybees (Apis mellifera) to odor from
PAP1-transgenic line 11 vs control flowers was evaluated using
72 naive foragers: the honeybee colony was kept inside a netted
enclosure (6 · 12 m2) so that the tested foragers were naive to
floral odors. Each bee was placed in a sectioned hollow plastic
tube and conditioned for PER according to standard procedures
(Shalit et al., 2004) as follows: the bee was fed 2 ll of a 50%
(w ⁄ v) sucrose solution and allowed to acclimate for 1.5 h before
the start of the trial. Odors were sampled from three control or
PAP1-transgenic flowers enclosed in 500-ml glass vials containing
one air inlet connected to a pump and an outlet connected to a
1-ml glass syringe. A constant computer-controlled stream of air
was pushed into the vial and the air stream exiting the vial from
the syringe flowed over the bee’s antennae and was immediately
vented into an exhaust vent behind the bee (Shalit et al., 2004).
Bees were subjected to two trials consisting of either GUS-
transgenic control or PAP1-transgenic odor samples. The order of
the odor sample presentation was: control ⁄ control, PAP1-transgenic ⁄
PAP1-transgenic, control ⁄ PAP1-transgenic or PAP1-transgenic ⁄
control (18 bees for each sample presentation order). Bees were
tested sequentially with an intertrial interval of 8 min. During a
trial, an odor was delivered for a period of 7 s. We recorded
whether the bee extended its proboscis during the first 4 s, before
delivery of an unconditioned stimulus (0.4 ll of a 50% sucrose
solution) with the last 3 s of odor delivery.
The bees’ differential responses were defined as ‘0’ or ‘1’ for
the same or different response, respectively, in the two trials. The
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binomial distribution of the bees’ differential responses to odors
from the two trials was determined, and the differential response
to either the same (control ⁄ control or PAP1-transgenic ⁄ PAP1-
transgenic) or different (control ⁄ PAP1-transgenic or PAP1-
transgenic ⁄ control) odors was analyzed by chi-squared test. In
addition, the differential responses to the same (control ⁄ control
or PAP1-transgenic ⁄ PAP1-transgenic) odors and the effect of the
order of the odor sample presentation (control ⁄ PAP1-transgenic
or PAP1-transgenic ⁄ control) were also analyzed using chi-
squared test. P < 0.05 was considered to be significant.
Statistical analysis
All statistical calculations were performed using JMP IN 5 (JMP
IN Software, SAS Institute Inc., Cary, NC, USA).
Results
PAP1-transgenic rose plants accumulate increased
anthocyanin levels
Transgenic rose plants cv Pariser Charme expressing cauliflower
mosaic virus (CaMV) 35S-driven PAP1 or uidA coding for the
GUS reporter gene (Fig. 1) were generated following agro-
inoculation of embryogenic callus (Dohm et al., 2001). The
transgenic nature of the PAP1 lines was confirmed using reverse
transcription PCR analyses: PAP1 expression was detected in
independent lines 6, 11, 12 and 13, but not in transgenic control
plants (Fig. 1d). PAP1-transgenic lines developed and matured
in a similar manner to their control counterparts, but demon-
strated increased pigmentation throughout plant development,
from the early plantlet stages in tissue culture to mature plants in
the glasshouse (Fig. 1a,b). PAP1-transgenic lines accumulated
six- to ninefold higher levels of anthocyanin per gram of leaf
tissue than the control transgenic plants (Fig. 1c).
PAP1 has previously been reported to exert broad transcrip-
tional activation of the phenylpropanoid pathway (Tohge et al.,
2005). Analysis of the transcriptional pattern of the anthocyanin
biosynthetic pathway in PAP1-transgenic rose lines revealed a
significant increase in transcript levels of both CHS and ANS
when compared with control plants (Fig. 2a,b). Transcript levels
of CHI, F3H and DFR were similar in both PAP1-transgenic and
control plants (Fig. 2c–e). RNA levels of PAL, catalyzing the first
committed step in the phenylpropanoid pathway, were also
comparable in PAP1-transgenic and control plants (Fig. 2f).
Flowers of PAP1-transgenic rose plants produce higher
levels of volatile compounds from phenylpropanoid and
isoprenoid biosynthetic pathways
PAP1-transgenic roses flowered normally in the glasshouse and
accumulated 2.5–5-fold higher levels of anthocyanins than
control GUS-transgenic flowers (Fig. 1c). We next examined
whether the effect of PAP1 extended to the production of volatile
secondary metabolites in independent PAP1-transgenic lines.
Qualitative and quantitative characterization of volatile
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(a) (b)
i ii iii i ii

iv v vi

iii iv
vii viii xi

(c) (d)

Fig. 1 Generation of Rosa hybrida cv Pariser Charme PAP1-transgenic plants. (a) Development of plantlets from somatic embryos in tissue culture:
b-glucuronidase (GUS)-transgenic control (i–iii), GUS-transgenic control following X-Gluc (5-bromo-4-chloro-3-indolyl b-D-glucuronic acid sodium salt)
staining (iv–vi) and PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1)-transgenic line 11 (vii–ix). (b) Leaves and flowers of GUS-transgenic control
(i, iii) and PAP1-transgenic line 11 (ii, iv). Insets in (i) and (iii) show X-Gluc-stained tissues. (c) Anthocyanin content in leaves and flowers of GUS-transgenic
control (Gus) and PAP1-transgenic lines 6, 11, 12 and 13 (Pap-6, Pap-11, Pap-12, Pap-13, respectively). Flowers were collected at stage 4 (sepals
beginning to retract, but petal whorl still closed). (d) Real-time PCR analysis of PAP1 transcript levels in seedlings of independent PAP1-transgenic lines.
Columns represent mean values (+ SE) of independent experiments (n = 3).

compounds accumulating in and emitted from flowers of
PAP1-transgenic rose lines 6, 11 and 12 was based on GC-MS
analyses conducted on floral tissue extracts and floral headspace
samples of detached flowers. The volatile profile obtained from
detached flowers has been shown to correspond to that obtained
from flowers growing intact on plants (Helsper et al., 1998). The
volatile composition of control and PAP1-transgenic flowers
consisted of compounds originating from the phenylpropanoid,
isoprenoid and fatty acid biosynthetic pathways (Table 1). The
level of the phenylpropanoid compound eugenol accumulated in
flowers of PAP1-transgenic lines was up to 20-fold higher than
that in flowers of control plants. The latter accumulated very low
levels of eugenol, representing c. 0.4% of the total internal pool
of volatile compounds, whereas, in PAP1 flowers, eugenol levels
reached 5–9% of the total pool of volatiles (Table 1). Levels of
all other phenylpropanoid compounds in the extract and head-
space, including that of the major accumulated compound benzyl
alcohol, were similar in PAP1-transgenic and control flowers.
Levels of the fatty acid volatile derivatives cis-3-hexenyl acetate
and hexanal were also similar in PAP1-transgenic and control
flowers (Table 1). Interestingly, an increase in the levels of emit-
ted volatiles of isoprenoid origin was revealed in flowers from all
PAP1-transgenic lines when compared with control flowers.
Specifically, levels of the major emitted terpenoid volatile com-
pound, the sesquiterpene germacrene D, were up to 8.5-fold
higher in PAP1-transgenic flowers. Emission of the norisopre-
noid compound b-ionone was also dramatically increased (up to
sixfold) in PAP1-transgenic flowers. By contrast, the internal pool
levels of detected terpenoid compounds were similar in
PAP1-transgenic and control flowers. Overall, the sum of emitted
volatile compounds was up to approximately fourfold higher in
flowers of PAP1-transgenic lines when compared with control
flowers, whereas the level of all accumulated volatile compounds
was not significantly different between the two (Table 1).
Increased levels of volatiles in PAP1-transgenic lines are
not caused solely by transcriptional activation of their
respective biosynthetic genes
Floral scent production presents highly ordered patterns and is
regulated at several levels. The transcriptional regulation of struc-
tural genes represents one of the main mechanisms controlling
volatile production (Verdonk et al., 2005; van Schie et al., 2006;
Colquhoun et al., 2010, 2011; Spitzer-Rimon et al., 2010). To
examine whether increased levels of volatile compounds
produced in PAP1-transgenic rose flowers stem from the tran-
scriptional up-regulation of their respective scent biosynthetic
genes, quantitative real-time PCR analyses were performed on
flowers just before anthesis (stage 4) when the machinery for
scent production is already active (Dudareva & Pichersky, 2006).

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(a) (b)

(c) (d)

(e) (f)

Fig. 2 Expression of anthocyanin biosynthetic genes. Quantitative real-time PCR analysis of (a) CHALCONE SYNTHASE (CHS), (b) ANTHOCYANIN
SYNTHASE (ANS), (c) CHALCONE ISOMERASE (CHI), (d) FLAVANONE 3-HYDROXYLASE (F3H), (e) DIHYDROFLAVONOL-4-REDUCTASE (DFR) and (f)
PHENYLALANINE AMMONIA LYASE (PAL) transcript levels in PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1)-transgenic lines 6, 11, 12 and 13
(Pap-6, Pap-11, Pap-12, Pap-13, respectively) when compared with b-glucuronidase (GUS)-transgenic control (Gus). Columns represent mean (+ SE)
values of independent experiments (n = 3). Significant differences between GUS control and PAP1-transgenic line: *, P < 0.05; calculated using Dunnett’s
method following one-way ANOVA, based on transcript level data normalized to ACTIN.

Transcript levels of GDS were significantly higher in all
PAP1-transgenic lines (Fig. 3a), in agreement with the emission
levels of germacrene D in independent PAP1-transgenic lines: the
highest levels of both transcript and emission were detected in
PAP1 line 12, followed by line 6 and line 11 (Fig. 3a, Table 1).
However, levels of CCD1 and EGS transcript were not correlated
with increased levels of their respective volatile products,
b-ionone and eugenol, among flowers of PAP1-transgenic lines:
transcript levels of EGS did not differ significantly between
PAP1-transgenic and control flowers, whereas a c. 10-fold reduc-
tion in CCD1 transcript level was observed in PAP1-transgenic
flowers when compared with controls (Table 1, Fig. 3b,c).
To further characterize the relationship between the levels of
volatile compounds and the transcripts of their respective struc-
tural genes, we analyzed the expression of several additional rose
scent biosynthetic genes. Transcript levels of OOMT1 (Lavid
et al., 2002), AAT1 (Shalit et al., 2003) and PAAS (Farhi et al.,
2009) were increased in flowers of PAP1-transgenic lines when
compared with controls (Fig. 3d–f). However, their respective
volatile products were below detection levels in both the
headspace and extracts of PAP1-transgenic and control flowers.
Phenylethyl alcohol, which has been shown to derive from
phenylacetaldehyde (Yang et al., 2009), was also not detected in
any of the examined flowers.
Humans and bees distinguish olfactory features of
PAP1-transgenic rose flowers
Floral scent has aesthetic value for humans and is of key impor-
tance to the plant in attracting pollinators (Smith et al., 2006;
Raguso, 2008). To examine whether the change in volatile profile
of PAP1-transgenic flowers is detectable to human scent percep-
tion, floral scent strength and preference were evaluated in
PAP1-transgenic vs control flowers using 2AFC tests (Fig. 4).
Irrespective of the order of sample presentation, PAP1-transgenic
floral samples (from all three independent lines) were given a
significantly higher score for scent strength than control flowers:
81%, 79% and 93% of the panelists indicated that PAP1 floral
samples from transgenic lines 6, 11 and 12, respectively, were
more strongly scented than floral samples from control plants

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Table 1 Volatile compounds accumulating in and emitted from PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1)- and b-glucuronidase
(GUS)-transgenic (control) Rosa hybrida cv Pariser Charme flowers

Amount

Compound Gus Pap-6 Pap-11 Pap-12

Phenylpropanoids
Eugenol 0.01 (± 0.002)p 0.22 (± 0.13)p* 0.13 (± 0.06)p* 0.22 (± 0.11)p*
Benzaldehyde 0.56 (± 0.08)e 0.38 (± 0.07)e 0.39 (± 0.19)e 0.49 (± 0.15)e
0.037 (± 0.001)p 0.036 (± 0.001)p 0.036 (± 0.001)p 0.047 (± 0.014)p
Benzyl alcohol 1.3 (± 0.23)p 1.44 (± 0.43)p 1.08 (± 0.24)p 0.89 (± 0.18)p

Sesquiterpenes
Germacrene D 0.48 (± 0.12)e 2.66 (± 0.41)e* 1.49 (± 0.72)e* 4.1 (± 0.92)e*
0.3 (± 0.11)p 0.45 (± 0.16)p 0.37 (± 0.14)p 0.3 (± 0.05)p
Unidentified 0.12 (± 0.01)e 0.64 (± 0.09)e* 0.47 (± 0.15)e* 1 (± 0.4)e*

Norisoprenoids
b-Ionone 0.06 (± 0.01)e 0.3 (± 0.06)e* 0.29 (± 0.11)e* 0.36 (± 0.08)e*
0.05 (± 0.0009)p 0.05 (± 0.001)p 0.07 (± 0.01)p 0.08 (± 0.01)p
Unidentified 0.05 (± 0.02)e 0.18 (± 0.1)e 0.39 (± 0.22)e* 0.05 (± 0.01)e
Unidentified 0.07 (± 0.02)p 0.12 (± 0.04)p 0.13 (± 0.03)p 0.15 (± 0.03)p *

Monoterpenes
Unidentified 0.22 (± 0.04)e 0.51 (± 0.04)e* 0.5 (± 0.14)e* 0.6 (± 0.04)e*
Unidentified 0.01 (± 0.0008)p 0.01 (± 0.002)p 0.01 (± 0.002)p 0.01 (± 0.003)p

Fatty acid derivatives

cis-3-Hexenyl acetate 0.08 (± 0.003)p 0.09 (± 0.019)p 0.1 (± 0.031)p 0.07 (± 0.007)p
Hexanal 0.51 (± 0.02)p 0.62 (± 0.02)p 0.53 (± 0.06)p 0.52 (± 0.03)p

Sum of emitted volatiles 1.49 (± 0.07) 4.66 (± 0.46)* 3.53 (± 0.93)* 6.59 (± 0.96)*
Sum of volatile internal pools 2.39 (± 0.32) 3.01 (± 0.69) 2.38 (± 0.4) 2.32 (± 0.54)

The levels of emission ((e), lg per flower per 24 h) and internal pools ((p), lg per flower) of volatile compounds produced by flowers of PAP1-transgenic
lines 6, 11 and 12 (Pap-6, Pap-11, Pap-12, respectively) when compared with GUS-transgenic control flowers (Gus) were analyzed quantitatively by gas
chromatography-mass spectrometry (GC-MS). Flowers at stage 7 (outer petal whorl is open, but inner petal whorl is still closed and reproductive organs
are not yet visible) were sampled. Values represent the means of three repeats; standard error is indicated in parentheses. Levels of emitted ⁄ accumulated
volatiles were compared between PAP1- and GUS-transgenic flowers by Student’s t-test following one-way ANOVA: significant difference from control
flowers *, P < 0.05. The identification of compounds was confirmed by comparison of mass spectra and retention times with those of authentic
compounds analyzed under similar conditions. The volatile compounds presented in the table represent 98% of the total detected volatiles.

(Fig. 4, top row, P < 0.05). In a separate set of trials designed to
test scent preference, significant preference for PAP1-transgenic
floral samples was revealed: 79%, 69% and 90% of the panelists
preferred the scent of floral samples from PAP1-transgenic lines
6, 11 and 12, respectively, to that of the control floral samples
(Fig. 4, bottom row, P < 0.05).
To examine whether PAP1-transgenic and control rose flower
odors could be distinguished by honeybees – the native pollina-
tors of some wild rose species (Shalit et al., 2004) – we moni-
tored the bees’ PER to PAP1-transgenic line 11 (which showed
the smallest increase in volatile emission levels) vs control flowers.
Bees were subjected to two trials consisting of the presentation of
control and PAP1-transgenic odor samples in one of the following
orders: control ⁄ control, PAP1-transgenic ⁄ PAP1-transgenic,
control ⁄ PAP1-transgenic or PAP1-transgenic ⁄ control. The bees’
differential responses were defined as ‘0’ or ‘1’ for same or differ-
ent responses, respectively, during the two trials. A significantly
higher differential PER (P < 0.0001) was revealed when bees
were subjected to odors from different (control ⁄ PAP1-transgenic
or PAP1-transgenic ⁄ control) floral samples when compared with
bees subjected to odors from the same (control ⁄ control or
PAP1-transgenic ⁄ PAP1-transgenic) floral samples (Fig. 5a).
Differential responses of bees subjected to odors from the combi-
nations of the same floral samples were very low and similar to
each other, indicating high consistency of the bees’ responses to
both floral odors (Fig. 5b). The bees’ ability to discriminate
between PAP1-transgenic and control flower odors was further
reflected by the significance of the effect of the order of odor pre-
sentation (control ⁄ PAP1-transgenic vs PAP1-transgenic ⁄ control)
on the differential PER: 0.38 vs 0.77 (P = 0.01) for control ⁄
PAP1-transgenic vs PAP1-transgenic ⁄ control sample order
presentation (Fig. 5c).
Discussion
PAP1 has been reported previously to exert transcriptional activa-
tion of the phenylpropanoid pathway, with overexpression of this
gene in several plant systems – Arabidopsis, tomato and Petunia –
leading to increased accumulation of anthocyanins (Borevitz
et al., 2000; Mathews et al., 2003; Matousek et al., 2006;
Ben Zvi et al., 2008a; Li et al., 2010; Shi & Xie, 2011).
Flowers of the latter also produce increased levels of volatile

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342 Research Phytologist

(a) (b)

(c) (d)

(e) (f)

Fig. 3 Expression of scent biosynthetic genes. Quantitative real-time PCR analysis of (a) GERMACRENE D SYNTHASE (GDS), (b) EUGENOL SYNTHASE
(EGS), (c) CAROTENOID CLEAVAGE DIOXYGENASE1 (CCD1), (d) ORCINOL O-METHYLTRANSFERASE1 (OOMT1), (e) GERANIOL ⁄ CITRONELLOL
ACETYL TRANSFERASE1 (AAT1) and (f) PHENYLACETALDEHYDE SYNTHASE (PAAS) transcript levels in flowers of PRODUCTION OF ANTHOCYANIN
PIGMENT1 (PAP1)-transgenic lines 6, 11 and 12 (Pap-6, Pap-11, Pap-12, respectively) when compared with b-glucuronidase (GUS)-transgenic control
(Gus) flowers. Rosa hybrida cv Pariser Charme flowers were collected at stage 4 (sepals beginning to retract, but petal whorl still closed) at 21:00 h.
Columns represent mean (+ SE) values of independent experiments (n = 3). Significant differences between GUS control and PAP1-transgenic line:
*, P < 0.05; calculated using Dunnett’s method following one-way ANOVA, based on transcript level data normalized to ACTIN.

phenylpropanoid compounds. The expression of PAP1 in the
rose ‘Pariser Charme’ enabled us to explore its effect on volatile
production in flowers that produce an array of volatile com-
pounds originating from diverse biochemical origins – sesquit-
erpenes, monoterpenes, norisoprenoids, fatty acid derivatives and
phenylpropanoid volatile compounds. This is in contrast with
petunia cv Blue Spark which produces exclusively phenylpropa-
noid volatile compounds (Ben Zvi et al., 2008a).
Flowers of mature PAP1-transgenic roses, similar to
PAP1-transgenic petunia (Matousek et al., 2006), produced
higher levels of anthocyanins than flowers from control plants.
Analysis of the volatile profile revealed increased levels of volatile
compounds from various biosynthetic origins in flowers of
PAP1-transgenic rose plants when compared with controls.
Specifically, PAP1-transgenic flowers accumulated higher levels
of the phenylpropanoid compound eugenol and emitted higher
levels of isoprenoid compounds. Increased emission of germac-
rene D in PAP1-transgenic flowers could be correlated with an
increased transcript level of GDS, suggesting that the enhanced
emission of germacrene D was caused by transcriptional activa-
tion of its structural gene. As the level of the internal pool of
germacrene D was similar in flowers of PAP1-transgenic and con-
trol plants, increased emission levels of germacrene D probably
resulted from release of the surplus produced as a result of the
increased generation of this compound in PAP1-transgenic
flowers. Similarly, analyses of germacrene D production in Rosa
hybrida ‘Fragrant Cloud’ revealed that its internal pool is
constant, and does not reflect the temporal diurnal changes in
germacrene D in the headspace of the flower (Hendel-Rahmanim
et al., 2007). Analyses of b-ionone levels revealed that, similar to
germacrene D, although the internal pool was not affected by
PAP1, the emitted levels were up to sixfold higher in
PAP1-transgenic vs control flowers. The native emission pattern
of b-ionone from flowers of rose, as well as petunia, has been
shown to correlate strongly with the transcript levels of CCD1,
encoding the enzyme that cleaves b-carotene to yield b-ionone
(Simkin et al., 2004; Huang et al., 2009). However, in
PAP1-transgenic roses producing increased levels of b-ionone,
the transcript level of CCD1 was strongly down-regulated. These
differences between metabolite and transcript levels in the trans-
genic background cannot be explained by the time of sampling
as, at a different time point (13:00 h), CCD1 transcript levels

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Fig. 4 Two-alternative forced choice scent trials. Floral scent strength and
preference trials were conducted on Rosa hybrida cv Pariser Charme
flowers of PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1)-
transgenic lines 6, 11 and 12 (Pap-6, Pap-11, Pap-12, respectively) when
compared with b-glucuronidase (GUS)-transgenic control flowers. Each
floral sample contained two open flowers at stage 7 (outer petal whorl is
open, but inner petal whorl is still closed and reproductive organs are not
yet visible). Untrained panelists (n = 24 per trial) were presented with
PAP1-transgenic and GUS-transgenic control flowers and asked to indicate
the sample with the stronger scent for the scent strength trials (top row) or
with the more appealing scent for the preference trials (bottom row). Trials
were conducted on three different occasions in a room with a constant
temperature of 24 ± 2C. Sectors in gray and white represent scores
for PAP1-transgenic and control flowers, respectively. The significance
(*, P < 0.05) of differences between odor samples of GUS control and
PAP1-transgenic flowers was analyzed by chi-squared test conducted
following the binomial distribution of the obtained scores.
were still similar in PAP1-transgenic and control flowers (not
shown). However, the discrepancy between metabolite and
transcript levels might be ascribable to differences in flux in the
isoprenoid pathway, similar to the previously described effects of
flux in the phenylpropanoid pathway on benzoid volatile produc-
tion (Zuker et al., 2002; Ben Zvi et al., 2008a; Colon et al.,
2010). Interestingly, as, in contrast with its product eugenol, the
transcript levels of EGS were not affected by PAP1, the increase
in this metabolite may also be explained by PAP1-enhanced flux
in the phenylpropanoid pathway (Ben Zvi et al., 2008a).
The complexity of floral scent was further evidenced by the
observation that the enhancement of PAAS, AAT1 and OOMT1
transcript levels in PAP1 transgenes did not yield increased levels
of their respective candidate volatile products (phenylacetalde-
hyde, geranyl ⁄ citronellyl acetates and orcinol methyl ether). The
lack of increase in the levels of these volatiles might be caused by
secondary modification of the metabolite, for example, the glyco-
sylation of phenylacetaldehyde ⁄ phenylethyl alcohol described in
rose flowers (Hayashi et al., 2004). However, the application of a
b-glucosidase mixture to PAP1-transgenic rose flowers did not
enhance volatile levels (data not shown). Similarly, in Petunia,
application of glycosidase did not affect volatile levels in
PAP1-expressing flowers (Ben Zvi et al., 2008a). Hence, the lack
of substrate availability may be a more plausible explanation for
the inability of PAP1 to enhance the production of the aforemen-
tioned metabolites. Indeed, the lack of available substrate has
been shown previously to be a limiting factor in volatile ester pro-
duction in various plant systems, including rose flowers (Shalit
et al., 2003; Beekwilder et al., 2004; Guterman et al., 2006).
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Research 343
(a) (b) (c)
Fig. 5 Differential response of bees to odors emitted from PAP1
(PRODUCTION OF ANTHOCYANIN PIGMENT1)-transgenic and
b-glucuronidase (GUS)-transgenic control Rosa hybrida cv Pariser Charme
flowers. The proboscis extension response was recorded from 72 naive
foragers subjected to flower odors of either controls (Gus) or
PAP1-transgenic line 11 (Pap-11). Bees were subjected to two trials
consisting of either odor emitted from three flowers. Eighteen bees were
used for each sample presentation order (Gus ⁄ Gus, Pap-11 ⁄ Pap-11,
Gus ⁄ Pap-11 or Pap-11 ⁄ Gus). The differential response of bees was
defined as ‘0’ or ‘1’ for the same or different response, respectively, during
the two trials. The binomial distribution of the differential responses of
bees to odor samples was analyzed by chi-squared test (significant differ-
ences; *, P < 0.05). (a) Mean differential response of bees to the same
(Gus ⁄ Gus and Pap-11 ⁄ Pap-11) vs different (Gus ⁄ Pap-11 or Pap-11 ⁄ Gus)
odors. (b) Differential response of bees to the same (Gus ⁄ Gus vs
Pap-11 ⁄ Pap-11) odors. (c) Differential response of bees to different
(Gus ⁄ Pap-11 vs Pap-11 ⁄ Gus) odors.
The changes in the volatile profile of PAP1-transgenic rose vs
control flowers detected by GC-MS analyses were also clearly
detectable by the olfactory analyses, revealing a strong preference
for the three analyzed PAP1-transgenic lines (as reported by
69–90% of panelists) over control flowers. This is one of a few
examples in which floral scent is up-regulated through genetic
transformation to levels that are detectable by untrained panelists
(Zuker et al., 2002; Lucker et al., 2004; Davidovich-Rikanati
et al., 2007). Interestingly, there was a direct correlation between
the levels of volatiles detected by GC-MS in the different lines,
that is, from highest to lowest in lines 12, 6 and 11, and the olfac-
tory scores given to these lines by the panelists.
Previous reports (Wright et al., 2002, 2005) have demon-
strated that bees can discriminate among flowers that vary in
scent intensity and ⁄ or relative concentrations of compounds.
Olfactory trials employing bees of PAP1 vs control rose flowers
revealed, for the first time, that the transgenic approach to floral
scent manipulation might yield changes in floral odor that are
sufficiently strong to be detectable by pollinators: bees condi-
tioned to the odor of either control or PAP1-transgenic flowers
were able to distinguish between the two. The bees’ ability to
discriminate between floral odors of PAP1-transgenic and control
plants was significantly higher when they were first presented
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344 Research
with, and conditioned to, the odor of PAP1-transgenic flowers,
further validating the notion that the change in odor level ⁄
composition is sufficient for discrimination by bees (Wright
et al., 2005; Smith et al., 2006; Reinhard et al., 2010).
The effect of PAP1 on volatiles from both the phenylpropa-
noid and isoprenoid pathways could be envisioned, although, to
date, investigations in planta have shown an exclusive association
of PAP1 with phenylpropanoid metabolism (Tohge et al., 2005).
For example, a comparison of PAP1-expressing Arabidopsis cells
with their wild-type counterparts revealed that the high levels of
anthocyanin accumulating in the former lead to the activation of an
array of genes from different metabolic pathways, not only phenyl-
propanoids (Shi & Xie, 2011). Furthermore, careful inspection of
the transcriptome of PAP1-overexpressing Arabidopsis plants (Dare
et al., 2008) using an annotated Arabidopsis gene dataset (Lange &
Ghassemian, 2003) revealed the transcriptional up-regulation of
several genes assigned to isoprenoid metabolism. Among these
were the functionally characterized genes coding for mevalonate
diphosphate decarboxylase (MPDC1; At2g38700) (Cordier et al.,
1999) and 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase
(MCT; At2g02500) (Rohdich et al., 2000). The enzymes MPDC1
and MCT are involved in very early stages of isoprenoid production
via mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate
(MEP) pathways, respectively. In addition, PAP1-transgenic
Arabidopsis seedlings accumulated increased transcript levels of ter-
pene synthase homolog (At3g32030) (Lange & Ghassemian,
2003; Dare et al., 2008). Interaction between different secondary
metabolic pathways, that is, phenylpropanoid and isoprenoid, was
also revealed in light signal transduction tomato mutants (Enfissi
et al., 2010), as well as in Impomoea flowers (Majetic et al., 2010)
and rice (Kim et al., 2010). The mechanism(s) underlying these
inter- and intra-relations in the metabolic pathways responsible for
the production of specialized metabolites remains to be deciphered.
Acknowledgements
This work was funded by Israel Science Foundation grant nos.
269 ⁄ 09 and 432 ⁄ 10 and BARD grant no. US-4322-10. A.V. is
an incumbent of the Wolfson Chair in Floriculture.
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