Biosynthesis of monoterpene scent compounds in roses

Article abstract of DOI:10.1126/science.aab0696

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.

Full text of DOI:10.1126/science.aab0696

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PLANT VOLATILES
plasma to more than 50 in eukaryotes (12). In
sequenced genomes of Arabidopsis, rice, and
grapevine, the number of genes coding for pu-
tative NUDX proteins is 28, 33, and 30, respectively
Biosynthesis of monoterpene scent
compounds in roses
(12, 13). RhNUDX1 shows the closest similarity
to AtNUDX1 (fig. S1). This protein was proposed
to have a similar function to Escherichia coli
mutator protein (MutT), which acts to eliminate
harmful compounds, such as 8-oxo–deoxyguanosine
triphosphate (8-oxo-dGTP), which may be mis-
incorporated in DNA during replication (14). We
have searched rose transcriptome database (15)
and identified 55 expressed sequence tags (ESTs)
corresponding to putative NUDX genes, indicat-
ing that, like in other species, NUDX1 belongs to
a gene family. All ESTs corresponding to RhNUDX1
showed high expression levels in fully opened
flowers (data S3). The other ESTs show no or weak
expression levels in blooming flowers.
1
1,2
1
Jean-Louis Magnard, Aymeric Roccia, Jean-Claude Caissard,
2
1
1
2
Philippe Vergne, Pulu Sun, Romain Hecquet, Annick Dubois,
3
1
1
Laurence Hibrand-Saint Oyant, Frédéric Jullien, Florence Nicolè,
2
4
5
5
Olivier Raymond, Stéphanie Huguet, Raymonde Baltenweck, Sophie Meyer,
5
3
6
3
Patricia Claudel, Julien Jeauffre, Michel Rohmer, Fabrice Foucher,
5
2
1
Philippe Hugueney, * Mohammed Bendahmane, * Sylvie Baudino *
The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules.
Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as
the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized
terpene synthases. Combining transcriptomic and genetic approaches, we show that
the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the
biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The
RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and
supports geraniol biosynthesis in planta.
In PM, RhNUDX1 was expressed in petals,
where scent is produced, with little to no expres-
sion in stamens, sepals, or young leaves (Fig. 2).
Expression increased at later stages of flower
development (Fig. 2B, stages 3 to 5) when scent
oses are used as ornamental plants in
gardens, as cut flowers, and as sources of
essential oils for perfume and cosmetics.
Breeding with a focus on cut flowers and
visual attributes can leave scent traits dis-
terpene biosynthesis in roses. We used cDNA
amplification fragment length polymorphism–
differential display (AFLP-DD) and DNA micro-
arrays to compare the transcriptomes of two rose
cultivars that have different scents (see supple-
mentary materials and methods).
R
advantaged (1). The cause for the lack of fra-
grance in these flowers is unknown and does
not seem to be linked to increased vase life (2).
Monoterpene alcohols and 2-phenylethanol char-
acterize typical rose scents; volatile phenolic com-
pounds characterize tea-scented roses (3). Although
genes involved in the biosynthesis of phenolic scent
compounds, 2-phenylethanol, and sesquiterpenes
have been characterized (4–6), the basis for mono-
terpene biosynthesis remains obscure.
The Papa Meilland (PM) cultivar emits a heavy
typical rose scent, mostly composed of mono-
terpene alcohols and 2-phenylethanol. The Rouge
Meilland (RM) cultivar produces very little scent
and only trace amounts of these compounds (table
S1). With AFLP-DD we identified two amplicons
favored in PM, one with homology to the Nudix
hydrolase family (DIF1) and one with homology
to a laccase protein (DIF38) (table S2). With mi-
croarrays we found 91 genes expressed more in
PM than in RM (data S1). The gene with the
highest differential expression (PM1, 7583-fold
increase in PM relative to RM) (table S2 and data
S1) also corresponded to the Nudix hydrolase.
We have named this gene RhNUDX1 (GenBank
accession number JQ820249). RhNUDX1 encodes
a 150–amino acid protein that contains the char-
acteristic Nudix domain (10) and is similar (59%
identity) to AtNUDX1 from Arabidopsis thaliana
In plants, geranyl diphosphate (GPP), precur-
sor of monoterpenes, is synthesized in plastids
from dimethylallyl diphosphate and isopentenyl
diphosphate supplied by the methylerythritol
Fig. 1. Correlation map of the expression of
RhNUDX1 and quantity of scent compounds
found in 10 rose cultivars. A nonparametric
Spearman correlation test was used. Strengths
of correlations are depicted by colors. Dark blue
squares with minus signs indicate a significant
negative correlation with correlation coefficient
r close to –1 (P < 0.05). Dark red squares with
plus signs indicate a significant positive correla-
tion with r close to +1 (P < 0.05). BAL, benzyl al-
cohol and benzaldehyde; CIT, citronellol; DMT,
3,5-dimethoxytoluene; EUG, eugenol and methyl-
eugenol; FAD, hexanal, E-2-hexenal, Z-3-hexenol,
E-2-hexenol, 1-hexanol, Z-3-hexenyl acetate, hexyl
acetate, and nonanal; FAR, E-a-farnesene, farnesol,
farnesal, and farnesyl acetate; GEM, germacrene
D, germacrene D-4-ol, and bicyclogermacrene; GER,
geraniol, geranial, geranic acid, and geranyl ace-
tate; ION, 3,4-dihydro-a-ionone and dihydro-a-ionol;
MON, b-myrcene, Z-b-ocimene, and E-b-ocimene;
NER, nerol and neral; PHE, 2-phenylethanol and
phenylacetaldehyde; SES, d-cadinene, elemol,
a-cadinol, t-cadinol, and t-muurolol; TMB, 1,3,5-
trimethoxybenzene.
4
-phosphate pathway (7). The monoterpenes in
essential oils are produced through the activity
of various monoterpene synthases (8). For exam-
ple, geraniol synthase (GES) converts GPP into
geraniol in basil (9). Here we investigate mono-
(
fig. S1). In a survey of 10 rose cultivars with con-
1
Laboratoire BVpam, EA3061, Université de Lyon/Saint-
trasting scent profiles (tables S1 and S2 and
data S2), RhNUDX1 expression correlated with
the presence of monoterpene alcohols (geraniol,
nerol, citronellol) and sesquiterpenes (farnesol,
farnesene, and farnesyl acetate) (Fig. 1). Nudix
hydrolases remove nucleoside diphosphates linked
to other moieties (10). Some may also accept non-
nucleoside substrates (11). These enzymes have
various functions and may act as diphosphoinosi-
tol polyphosphate phosphohydrolases, coenzyme A
pyrophosphatases, adenosine diphosphate–ribose
pyrophosphatases, diadenosine polyphosphate
hydrolases, and mRNA decapping enzymes (fig.
S1B). Nudix hydrolyses are found in animals, plants,
and bacteria. The number of Nudix representa-
tives in each species varies from one in Myco-
Etienne, 23 Rue du Dr Michelon, F-42000, Saint-Etienne,
France. Laboratoire Reproduction et Développement des
Plantes UMR Institut National de la Recherche Agronomique
(
Supérieure de Lyon, 46 Allée d’Italie, 69364 Lyon Cedex 07,
France. INRA, Institut de Recherche en Horticulture et
Semences (INRA, AGROCAMPUS-OUEST, Université
d’Angers), SFR 4207 QUASAV, BP 60057, 49071 Beaucouzé
Cedex, France. Génomiques Fonctionnelles d’Arabidopsis,
Unité de Recherche en Génomique Végétale, UMR INRA
1
France. INRA, Université de Strasbourg, UMR 1131 Santé
de la Vigne et Qualité du Vin, 28 Rue de Herrlisheim, F-68000
Colmar, France. Université de Strasbourg–CNRS, UMR 7177,
Institut Le Bel, 4 Rue Blaise Pascal, 67070 Strasbourg Cedex,
France.
*
2
INRA)–CNRS, Université Lyon 1-ENSL, Ecole Normale
3
4
165–Université d’Evry Val d’Essonne–ERL CNRS 8196, Evry,
5
6
Corresponding author. E-mail: sylvie.baudino@univ-st-etienne.
fr (S.B.); philippe.hugueney@colmar.inra.fr (P.H.); mohammed.
bendahmane@ens-lyon.fr (M.B.)
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emission is at its maximum (16). RhNUDX1 pro-
tein accumulated at stage 4 in PM petals at the
predicted molecular mass of 16.8 kD, but not in
petals of the scentless cultivar RM (Fig. 2C).
RhNUDX1 seems to carry no transit peptide, as
evidenced by its sequence (17) and by cytosolic
localization after transient expression in Nicoti-
ana benthamiana of RhNUDX1 protein fused to
green fluorescent protein (GFP) at its N or C ter-
minus (fig. S2).
Fig. 2. Analysis of RhNUDX1 expression in Rosa
x hybrida. Flower developmental stages were de-
fined as in (16). SE values are indicated by vertical
bars (n = 6 replicates). Means with different let-
ters (a, b, c) are significantly different (Tukey’s HSD
test, P < 0.05). (A) Expression in young leaves and
floral organs at stage 4 (S4), analyzed by quantitative
polymerase chain reaction (qPCR). (B) Expression in
petals during development (from S1 to S5), analyzed
by qPCR. (C) Western blot analysis of RhNUDX1
protein in Rosa x hybrida petals. M, Molecular weight
marker; R, recombinant protein produced in E. coli.
Expression of RhNUDX1 correlated with ter-
pene biosynthesis. When we used an RNAi con-
struct to knock down expression of RhNUDX1 in
Rosa chinensis cv. Old Blush (OB), a rose that
produces high levels of the monoterpene gera-
niol, three independent transgenic events were
generated, one of which (line A) showed reduced
RhNUDX1 expression (Fig. 3A). Volatile mono-
terpenes geraniol and geranial from petals were
positively correlated with RhNUDX1 expres-
sion levels (Fig. 3B and table S3). Line A had the
least monoterpene content. Amounts of other
volatiles were unaffected (fig. S3). To confirm
the consequences of RhNUDX1 down-regulation,
we used a transient transformation strategy,
which has been shown to allow efficient gene
silencing in floral tissues (18). Agrobacterium-
mediated transient transformation of rose petals
was found to be extremely genotype-dependent.
The best transformation efficiencies were ob-
tained using the heavily scented genotype known
as The Mac Cartney Rose, as shown by the expres-
sion of GFP (fig. S4). These petals were infiltrated
with Agrobacterium harboring the RhNUDX1
RNA interference (RNAi) construct used for sta-
ble transformation. Compared with petals expres-
sing GFP, petals expressing the RhNUDX1 RNAi
construct had fewer monoterpenes; other classes
of scent compounds were not significantly af-
fected (fig. S5 and table S4).
To study the genetic basis of monoterpene
biosynthesis in roses, we analyzed an F1 progeny
from crosses between genotypes with different
scent profiles. This mapping population con-
sists of a full-sib family of 116 hybrids derived
from a diploid cross between OB and a hybrid Rosa
wichurana (RW) originating from the Bagatelle
garden (Paris, France). The female parent (OB)
produced high amounts of geraniol, whereas the
male parent (RW) did not. RhNUDX1 was ex-
pressed~4000 timesmoreinOBthaninRW(fig.S6).
Gas chromatography–mass spectrometry (GC-MS)
analyses of petal volatiles showed that the ge-
raniol content segregated in the progeny (fig.
S7C). We detected a major quantitative trait locus
Fig. 3. Characterization of RNAi-RhNUDX1 transgenic rose lines. (A) Real-time quantitative reverse
transcription PCR analyses of RhNUDX1 expression in petals of nontransformed plants, in plants
transformed with the Gus reporter gene under the control of the 35S promoter (35S::GUS), and in three
transgenic RNAi-RhNUDX1 lines. SE values are indicated by vertical bars (n = 6 replicates). (B) GC-MS
analyses of the petal volatile monoterpenes in RNAi-RhNUDX1 transgenic rose lines. Amounts are
expressed in micrograms per gram of fresh leaf weight (mg/g FW). SE values are indicated by vertical
bars (n = 8 to 12 replicates). For both panels, means with different letters (a and b) are significantly
different (Tukey’s HSD test, P < 0.05).
failed to yield geraniol or farnesol after incuba-
tion with GPP and farnesyl diphosphate (FPP),
suggesting that RhNUDX1 lacks terpene syn-
thase activity (fig. S8). Recombinant RhNUDX1
(Fig. 4B) showed diphosphohydrolase activity
when incubated with GPP and FPP. The pro-
ducts were geranyl monophosphate (GP) and
farnesyl monophosphate (FP), respectively (Fig.
4A, fig. S9, and table S5). Optimal activity enzy-
matic activity occurred around pH 8, with very
low activity below pH 6 (fig. S10). RhNUDX1
m
S5). The K for GPP was two orders of magnitude
(QTL) for geraniol biosynthesis on the female
lower than that of GES (9) and in the same range
as that of the MutT nudix protein of E. coli
with its substrate 8-oxo-dGTP (14). Conversely,
RhNUDX1 exhibited poor activity with 8-oxo-
dGTP and dGTP.
To investigate RhNUDX1 involvement in ge-
raniol biosynthesis, we compared its activity to
that of GES from basil (9). We used Agrobacterium-
mediated transient expression to express both in
N. benthamiana. We verified that the RhNUDX1
protein was accumulated in tobacco leaves (fig.
S11). Expression of GES in plastids resulted in the
linkage group 2 (LG2). This QTL explained 76% of
the observed variation in geraniol content (LOD
score 39) (fig. S7B). When mapped on the female
LG2, RhNUDX1 colocalized with this QTL for ge-
raniol production (fig. S7A). Together with genetic
colocalization, the transgenic down-regulation data
supports a role of RhNUDX1 in geraniol biosyn-
thesis in roses.
Our hypothesis is that the RhNUDX1 protein,
located in the cytosol, is in the biosynthetic path-
way for monoterpenes. Recombinant RhNUDX1
m
exhibited low Michaelis constant (K ) values for
GPP and FPP (140 and 480 nM, respectively) (table
8
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Fig. 4. Functional characterization of RhNUDX1
in vitro and in planta. (A) Decrease in GPP and
concomitant increase in GP after incubation with
recombinant NusA-RhNUDX1 fusion protein. NusA-
RhNUDX1 (250 ng) was incubated in the pres-
ence of 1 mM GPP at 30°C. At the indicated time
points, GPP and GP were quantified by liquid
chromatography–mass spectrometry (LC-MS). (B)
SDS–polyacrylamide gel electrophoresis analysis
of recombinant NusA-RhNUDX1 protein. 1, molec-
ular weight marker; 2, soluble protein extract from
NusA-RhNUDX1–expressing E. coli; 3 and 4, puri-
fied NusA-RhNUDX1 protein. Molecular weights
(in kilodaltons) are indicated. (C) Accumulation
of geranyl glycosides after transient expression
of RhNUDX1 in N. benthamiana. GES from basil (9) was expressed as a full-length protein including its transit peptide (plastidic GES, 35S::plaGES) and as a
truncated protein without transit peptide (cytosolic GES, 35S::cytGES). This latter construct was aimed at comparing GES and RhNUDX1 activity in a
cytosolic context. GFP was used as a control.Geranyl glycosides were quantified by LC-MS 96 hours after transformation. Amounts are expressed in micrograms per
gram of fresh leaf weight (mg/g FW) of geranyl glucoside equivalent, as means of triplicate assays. Bars indicate SE.
Fig. 5. Hypothetical biogenetic scheme for the formation of
free geraniol in rose petals. 1, dimethylallyl diphosphate; 2,
isopentenyl diphosphate; 3, GPP; 4, GP; 5, geraniol.
release of geraniol (fig. S12) and the accumula-
tion of geranyl glycosides (Gglyc) (Fig. 4C). Ex-
pression of GES or RhNUDX1 in the cytosol both
resulted in similar accumulations of Gglyc and
geraniol (Fig. 4 and fig. S12). As RhNUDX1 did
not dephosphorylate GP to geraniol in vitro (fig.
S8), we assumed that enzymes (presumably phos-
phatases) carried out that conversion in planta.
Indeed, we incubated rose petal proteins with
GP and found that it was efficiently converted to
geraniol (fig. S13). Although the RhNUDX1 en-
zyme was able to use FPP as a substrate in vitro,
farnesol or farnesol glycoside did not accumulate
in RhNUDX1-expressing tissues. Altogether, our
data show that RhNUDX1 supports efficient ge-
raniol and Gglyc biosynthesis in planta and
that, in a cytosolic context, RhNUDX1 is equiv-
alent to GES.
As GPP is a substrate for RhNUDX1, which is
itself localized in the cytoplasm, we ask about the
origin of GPP. GPP is a precursor for mono-
terpenes through pathways generally localized in
plastids. Only a few studies suggest that GPP
could be generated by the mevalonate pathway
in some plants like rose and other Rosaceae (19–22).
GPP for use in the cytosol for the biosynthesis of
monoterpenes may be exported from plastids (23)
or could also be a by-product of FPP synthase
activity (24) or may be produced by a cytosolic GPP
synthase.
monoterpenes it produces, and our finding may
provide leverage to restore scent in roses with
low levels of RhNUDX1 expression.
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SUPPLEMENTARY MATERIALS
(
www.sciencemag.org/content/349/6243/81/suppl/DC1
Material and Methods
Figs. S1 to S13
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6 March 2015; accepted 29 May 2015
10.1126/science.aab0696
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3 JULY 2015 • VOL 349 ISSUE 6243 83

Biosynthesis of monoterpene scent compounds in roses
Jean-Louis Magnard et al.
Science 349, 81 (2015);
DOI: 10.1126/science.aab0696
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