Download Role of Petal-Specific Orcinol O-Methyltransferases

Role of Petal-Specific Orcinol O-Methyltransferases in
the Evolution of Rose Scent1
Gabriel Scalliet2, Claire Lionnet, Mickaël Le Bechec3, Laurence Dutron, Jean-Louis Magnard,
Sylvie Baudino, Véronique Bergougnoux, Frédéric Jullien, Pierre Chambrier, Philippe Vergne,
Christian Dumas, J. Mark Cock, and Philippe Hugueney*
Laboratoire Reproduction et Développement des Plantes, Unité Mixte de Recherche 5667 Centre National
de la Recherche Scientifique-Institut National de la Recherche Agronomique-Ecole Normale Supérieure de
Lyon-Université Claude Bernard Lyon 1, IFR128 Biosciences Lyon-Gerland (G.S., C.L., P.C., P.V., C.D., P.H.),
and Département des Sciences de la Vie et de la Terre (M.L.B., L.D.), Ecole Normale Supérieure de Lyon,
69364 Lyon, France; Laboratoire de Biotechnologies Végétales Appliquées aux Plantes Aromatiques et
Médicinales, Université Jean Monnet, 42023 Saint-Etienne, France (J.L.M., S.B., V.B., F.J.); and
Unité Mixte de Recherche 7139 Centre National de la Recherche Scientifique-Université Pierre et Marie
Curie, Végétaux Marins et Biomolécules, 29682 Roscoff, France (J.M.C.)
Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic
methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are
descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former.
Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing
varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these
cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells’ secretory machinery. OOMT gene sequences
were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these
varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression
may have been a critical step in the evolution of scent production in roses.
The past 10 years have seen rapid progress in flower
scent research, the initial breakthrough coming from
the pioneering work on (S)-linalool synthase from
flowers of Clarkia breweri (Pichersky et al., 1995). The
purification of (S)-linalool synthase provided amino
acid sequence information that allowed the isolation of
the corresponding gene (Dudareva et al., 1996). The
characterization of additional genes involved in the
biosynthesis of floral volatiles in C. breweri followed,
including those encoding enzymes for the synthesis of
the phenylpropanoids methyl eugenol and methyl
1
This work was supported by the Région Rhône-Alpes (France),
the Institut National de la Recherche Agronomique, and the Centre
National de la Recherche Scientifique.
2
Present address: Syngenta Crop Protection Münschwilen AG,
WST–540.2.51 Schaffhauserstrasse CH–4332 Stein, Switzerland.
3
Present address: Unité Mixte de Recherche Centre National de la
Recherche Scientifique 6204, Faculté des Sciences et des Techniques,
2 rue de la Houssinière, 44322 Nantes cedex 3, France.
* Corresponding author; e-mail [email protected];
fax 33–4–72728600.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Philippe Hugueney ([email protected]).
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.070961.
18
isoeugenol (Wang et al., 1997), benzyl acetate (Dudareva
et al., 1998), and methyl salicylate (Ross et al., 1999).
More recently, a wealth of information on the biosynthesis of floral volatiles has come from studies of
the common garden snapdragon (Antirrhinum majus;
Dudareva and Pichersky, 2000; Dudareva et al., 2000,
2003; Tholl et al., 2004). Until very recently, however,
C. breweri and snapdragon represented the only plant
species in which isolation of enzymes and genes
responsible for the formation of scent volatiles in the
flower had been accomplished (Dudareva and Pichersky,
2000). Over the past three years, this situation has
begun to change, and several investigations have been
launched with the aim of understanding the biosynthesis of floral volatiles in other plant models, including Arabidopsis (Arabidopsis thaliana; Aharoni
et al., 2003; Chen et al., 2003), petunia (Petunia hybrida;
Verdonk et al., 2003, 2005; Boatright et al., 2004), and
the rose (Rosa spp.). Genomic approaches have recently been used to analyze the transcriptome of rose
petals, leading to the identification of several genes
potentially involved in scent production (Channelière
et al., 2002; Guterman et al., 2002). Functional characterization of some of these genes led to the identification of a sesquiterpene synthase involved in the
production of germacrene D (Guterman et al., 2002),
an alcohol acetyltransferase involved in the formation
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Biosynthesis of Scent in Rose Petals
of geranyl acetate (Shalit et al., 2003), and orcinol
O-methyltransferases (OOMTs). OOMTs, encoded
by two closely related OOMT genes, OOMT1 and
OOMT2, catalyze the last two steps of the biosynthetic
pathway leading to 3,5-dimethoxytoluene (DMT), the
major scent compound in many rose varieties (Lavid
et al., 2002; Scalliet et al., 2002). In Rosa chinensis,
OOMTs are also able to catalyze the final steps of an
alternative pathway leading from phloroglucinol to
the trimethylated molecule 1,3,5-trimethoxybenzene
(TMB; Scalliet et al., 2002). In addition to OOMTs, TMB
biosynthesis involves a phloroglucinol O-methyltransferase, which was characterized recently (Wu et al.,
2004).
Nowadays, phenolic methyl ethers are emitted by
flowers from most modern rose varieties (Flament
et al., 1993). However, the progenitors of modern roses
included both European species (e.g. Rosa gallica, Rosa
phoenicia, and Rosa moschata) and Chinese species (e.g.
R. chinensis and Rosa gigantea), and, interestingly, emission of phenolic methyl ethers was originally restricted to Chinese rose species and was not found in
the European species (Nakamura, 1987; Flament et al.,
1993; Joichi et al., 2005). In this study we have developed methods to study OOMT expression and
activity at the organ, cellular, and subcellular levels
in a range of rose varieties. We show that European
roses possess OOMT-like sequences in their genomes
but do not express OOMT activity in their petals,
providing a molecular explication for the absence of
methylated phenolic compounds in the scent of these
roses.
RESULTS
DMT Production Is Correlated with the Presence
of OOMT Protein and Enzyme Activity
To investigate the reasons for the absence of methylated phenolic compound emission by European rose
flowers, OOMT activity was measured in petals of two
typical European roses, R. gallica and Damask rose
(Rosa damascena). R. gallica is a wild European species
which, together with two other European species, is
a progenitor of Damask, a rose variety used for attar
production (Iwata et al., 2000). Both R. gallica and
Damask rose possess a typical European scent, rich in
monoterpenes and phenylethanol but devoid of phenolic methyl ethers (Flament et al., 1993; Oka et al.,
1999). In contrast to R. chinensis cv Old Blush, no
significant OOMT activity could be detected when R.
gallica and Damask rose cell-free petal extracts were
incubated with orcinol and S-adenosyl-L-[methyl14
C]Met (Fig. 1). These data suggest that the European
species do not synthesize DMT because they lack
active OOMT enzymes.
To investigate this further, a polyclonal antibody
was raised against the OOMT1 protein from the rose
variety Old Blush, expressed in Escherichia coli as a
Figure 1. Absence of OOMT activity in European roses. TLC analysis of
OOMT activity in European and Chinese rose petal extracts (20 mg
of total protein), incubated with orcinol (1 mM) in the presence of
S-adenosyl-L-[methyl-14C]Met (50 mM). R.g., R. gallica; R.d., Damask
rose; R.c., R. chinensis cv Old Blush. Arrows indicate the positions of
the origin (Ori) and the reaction product MHT.
glutathione S-transferase-fusion protein, as described
by Scalliet et al. (2002). After cleavage of the glutathione S-transferase moiety, purified recombinant OOMT1
was used to immunize rabbits. Note that this antibody
recognizes both OOMT1 and the closely related OOMT2
protein, but not the more distantly related caffeic acid
O-methyltransferase (accession no. AJ439740; data not
shown).
We first used the anti-OOMT antibody to determine
the abundance of OOMT proteins in different tissues
from R. chinensis cv Old Blush, including petals from
flowers at different developmental stages (stages 3, 4,
and 5, according to Guterman et al., 2002), three other
floral organs (sepals, stamens, and pistils) from flowers at stage 3, and young leaves collected from growing branches. Western-blot analysis of these samples
showed that OOMTs accumulated specifically in petals (Fig. 2A). OOMT accumulation was developmentally
regulated, reaching a maximum in the open flower
(stage 4). OOMTs were not detected in other parts of
the flower (pistil, stamen, and sepals) or in leaves. This
petal-specific pattern of accumulation of OOMTs was
consistent with previous observations of OOMT transcript abundance, with the exception that significant
levels of OOMT mRNA had also been detected in Old
Blush stamens (Scalliet et al., 2002).
Having established the pattern of accumulation of
OOMT protein in the TMB-producing variety Old
Blush, we used the same antibody to assay for OOMT
protein in the European species R. gallica and Damask
rose. No signal corresponding to putative OOMT
proteins was detected following western-blot analysis
of protein extracts from a range of different tissues
including petals at stage 4, where the highest levels of
protein were detected in the Old Blush samples (Fig.
2B). The absence of phenolic methyl ethers in the scent
of the European species R. gallica and Damask rose was
therefore correlated with an absence of both OOMT
protein and enzyme activity.
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Scalliet et al.
the use of green fluorescent protein (GFP) fusion
proteins to investigate the subcellular localization of
OOMTs in rose petal epidermal cells.
Biolistic Transformation of Rose Petal Secretory Cells and
Subcellular Localization of OOMT-GFP Fusion Proteins
Remarkable work using plant secretory cell model
systems, such as peppermint (Mentha piperita) glandular
Figure 2. Western-blot analysis of OOMT expression. A, Western-blot
analysis of OOMT expression in different organs of R. chinensis cv Old
Blush. L, Leaves; Se, sepals; P3, petals at stage 3; P4, petals at stage 4;
P5, petals at stage 5; St, stamens; Pi, pistils. Each lane was loaded with
10 mg of total protein. B, Western-blot analysis of OOMT expression in
European roses. Total protein extracts from flower organs and leaves of
Damask rose and R. gallica were analyzed by western blot, using antiOOMT antibody. Old Blush (OB) petals were used as positive control.
L, Leaves; Se, sepals; P, petals (stage 4); St/Pi, stamens 1 pistils. Each
lane was loaded with 10 mg of total protein.
OOMTs Immunolocalize Predominantly to the Rose
Petal Epidermis
Rose petal epidermal cells are believed to be the
major site of scent production in roses (Stubbs and
Francis, 1971; Loomis and Croteau, 1973). However,
there is no direct evidence that scent biosynthesis enzymes are located in these cells. The experiments
described above showed that OOMT proteins were
accumulated specifically in petals of R. chinensis cv Old
Blush. To determine the tissue localization of phenolic
methyl ethers production, OOMT distribution in rose
petals was investigated by immunolocalization. Immunolocalization experiments were carried out using
the modern Tea rose Rosa x hybrida cv Lady Hillingdon
because this rose has been shown to produce large
amounts of DMT (Nakamura, 1987; Joichi et al., 2005)
and its petals proved to be well suited to microscopy
techniques. Cross sections of petals from Lady
Hillingdon were incubated with the anti-OOMT antibody and OOMTs were visualized using secondary
antibodies coupled to alkaline phosphatase and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl
phosphate (BCIP) as substrate. This analysis indicated
that OOMTs were localized principally in the conical
cells that constitute the upper epidermis of the petal
and, to a lesser extent, in the subepidermal cell layer
(Fig. 3, A and B). Weaker staining was also detected in
the lower epidermis, indicating the presence of a lower
level of OOMTs in these cells. No signal was observed
when the petal sections were incubated with preimmune serum from the same rabbit (Fig. 3C).
The observation that OOMTs are located predominantly in the petal epidermis supports the hypothesis
that this cell layer plays an important role in scent
production. Moreover, the identification of an enzyme
involved in scent production in these cell types opens
up the possibility of investigating scent biosynthesis at
the cellular level. As a first step toward this objective,
we developed an in vivo imaging strategy based on
Figure 3. Tissue and cellular localization of OOMT proteins. A to C,
Immunolocalization of OOMTs in Lady Hillingdon petals. A, Cross
sections of young petals (stage 3) were incubated with anti-OOMT
polyclonal antibody. B, Higher magnification of adaxial epidermis cells
from A. C, Control incubated with preimmune serum. OOMTs were
visualized using goat anti-rabbit secondary antibodies coupled to
alkaline phosphatase and NBT/BCIP as substrate. D, Ultrastructure of
a rose petal adaxial epidermal cell in transverse section (transmission
electron microscopy). E to L, Transient expression of GFP fusion
proteins following biolistic transformation of rose petal epidermal
cells. E to G, Optical sections in a rose epidermal cell expressing GFP.
E, Projection of 10 3 1 mm Z-series optical sections through the apex of
the conical cell. F, One micrometer optical section through the middle
of the same cell. G, One micrometer optical section through the base of
the same cell. H, Expression of GFP targeted to plastids (projection of
25 3 1 mm optical sections). I, Expression of GFP targeted to the ER
(projection of 25 3 1 mm optical sections). J, Expression of GFP-OOMT
fusion protein (projection of 10 3 1 mm optical sections through the
apex of the conical cell). K, Expression of OOMT-GFP fusion protein,
single 1 mm optical section through the labeled structure at the apex of
the cell. L, Projection of 25 3 1 mm optical sections of the basal region
of another cell expressing OOMT-GFP fusion protein. Bars 5 10 mm.
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Biosynthesis of Scent in Rose Petals
trichomes, has provided a greatly improved understanding of the metabolism of monoterpenes (Mahmoud
and Croteau, 2002). However, because of the dispersed
distribution of secretory structures on the organ surface, currently used secretory model systems are not
well suited to a combined use of transient expression
from gene gun delivered constructs and in vivo imaging techniques. On the other hand, biolistic transformation of petal cells has been limited to approaches
such as promoter characterization (Clark and Sims,
1994; Quattrocchio et al., 1999).
The rose petal epidermis provides an ideal target for
biolistic delivery of transgene constructs because it
consists of a homogenous layer of secretory cells. We
found that petal epidermal cells of several rose varieties, including Old Blush and Lady Hillingdon, could
be used for this type of experiment. However, petals of
the cut flower variety R. x hybrida cv Anna (PEKcougel)
gave the best results because flowers can be obtained
regularly throughout the year under greenhouse conditions and they have a long vase life of about 9 d due to
their very resistant petals. Moreover, petals of this variety
emit large amounts of DMT (data not shown).
To provide a framework for the analysis of gene
expression in petal epidermal cells, the internal structure of these cells was determined by transmission
electron microscopy of tangential cross sections of
Anna petals. Figure 3D shows a cross section in the
apical region of a petal secretory cell. The cell is surrounded by a thick cell wall, covered by a waxy cuticle, with undulations that are due to the characteristic
striated aspect of these cells (Stubbs and Francis, 1971).
The dense cytoplasm contains starch-rich plastids and
many vacuolar structures. These vacuoles contain variable amounts of electron-opaque material, as previously observed in petal epidermal cells of other flowers
(Weston and Pyke, 1999).
Transformed epidermal cells accumulated large
amounts of GFP or GFP fusion proteins, leading to
an intense fluorescence 24 h (for GFP alone) or 48 h (for
GFP fusion proteins) after transformation. Epidermal
cells of excised rose petals remained metabolically
active and were able to synthesize chimeric proteins
over a period of several days. This is consistent with
previous studies, which have shown that rose flowers
can maintain scent biosynthesis without an external
carbohydrate supply (Helsper et al., 1998). Metabolism
in excised rose petals presumably utilizes the large
stock of starch that accumulates during the early steps
of petal development (Fig. 3D).
Following biolistic transformation, the cellular localizations of GFP fusion proteins were studied using
confocal laser scanning microscopy. Initially, a number
of markers were used to characterize different compartments of the rose petal secretory cell (Fig. 3, E–I).
Expression of nontargeted GFP in Anna petal epidermis cells confirmed that these cells are extremely
vacuolized, with a central vacuole surrounded by
numerous vacuole-like compartments (Fig. 3, E–G).
Plastids were visualized using plastid-targeted GFP
(Fig. 3H), and the expression of m-GFP5-HDEL
(Haseloff et al., 1997) gave the reticulated pattern of
GFP localization typical of endoplasmic reticulum
(ER) in plant cells (Fig. 3I; Boevink et al., 1998).
To investigate the subcellular localization of OOMT,
constructs encoding GFP fused either to the N terminus (GFP-OOMT) or to the C terminus (OOMT-GFP)
of OOMT1 from Old Blush were introduced into petal
epidermal cells by biolistic transformation. Anna petal
cells expressing GFP-OOMT fusion protein showed a
fluorescence pattern very similar to that obtained with
nontargeted GFP, indicating that GFP-OOMT behaved
as a soluble cytosolic protein (Fig. 3J). In contrast,
OOMT-GFP fusion protein was restricted to particular
regions of the cell, giving rise to intense fluorescent
signals (Fig. 3K). The same kind of pattern was obtained when OOMT-GFP was expressed in petal
epidermis cells of other rose varieties, such as Lady
Hillingdon (Fig. 3L) or Old Blush (data not shown).
These intensely fluorescing regions were often localized in the apical region of the cells (Fig. 3L) but were
also found sometimes in the basal region (Fig. 3K).
Based on the predominantly membrane-associated
localization of the endogenous OOMT enzymes at
this stage of development (see next paragraph), we
propose that the localization of the OOMT-GFP fusion
protein is most likely to reflect that of the endogenous
protein. This highly localized distribution was of
particular interest because OOMTs catalyze the last
steps of DMT biosynthesis and these enzymes might
therefore be expected to be associated with a putative
structure that would mediate DMT secretion.
A Fraction of the OOMT Protein Is Membrane Bound
in Rose Petal Cells
We were not able to investigate the subcellular localization of the GFP fusion proteins in rose petals cells
using ultracentrifugation experiments because of the
limited number of transformed cells obtained using
biolistic techniques. However, ultracentrifugation experiments were used to investigate the localization of
the endogenous OOMTs. Rose petals were collected at
different developmental stages (stages 3, 4, and 5) and
cell-free extracts of these petals were subjected to ultracentrifugation (150,000g for 1 h). Supernatants and
pellets were analyzed by western blot, using antiOOMT antibodies (Fig. 4). Rose OOMTs were detected
in both the supernatant and in the pellet fractions after
centrifugation at 150,000g, with the relative amounts of
the soluble and the membrane-bound forms varying
depending on the developmental stage. In young petals
(stage 3) the majority of OOMT protein was detected
in the 150,000g supernatant, a weaker signal being
detected in the pellet. The proportion of membranebound OOMT increased in petals from flowers at stage
4 and finally reached 100% at stage 5, no soluble
OOMT being detected at this stage (Fig. 4A). To test
how tightly OOMTs were associated with membranes,
microsomes were prepared from petals sampled at
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Scalliet et al.
Figure 4. Association of OOMT with rose petal microsomes. A, R. x
hybrida cv Anna petals were sampled at development stages 3, 4, and
5. Cell-free extracts (C) from these petals were centrifuged at 150,000g
for 1 h and supernatants (S) and pellets (P) were analyzed by westernblot using the anti-OOMT antibody. B, Microsomal membranes, prepared from petals at stage 4, were incubated for 30 min with buffer (1),
2 M NaCl (2), 0.1% Triton X-100 (3), 0.1 M Na2CO3 (4), 0.1 M NaOH (5),
or 6.8 M urea (6). The membranes were recovered by a centrifugation at
150,000g for 1 h and analyzed by western blot using anti-OOMT
antibody.
stage 4 and incubated in the presence of either 2 M
NaCl, 0.1% Triton X-100, 0.1 M Na2CO3, 0.1 M NaOH, or
6.8 M urea (Fig. 4B). OOMTs were released from the
membranes by alkaline treatments such as Na2CO3
and NaOH incubations, but not by any of the other
reagents, indicating that the microsome-associated
OOMTs were firmly bound to membranes. To summarize, native OOMTs in rose petals were detected in
both the soluble fraction and tightly bound to microsomes. The membrane-bound fraction was present
throughout petal development but increased in relative abundance during maturation. Based on these
observations, it is probable that the highly localized
expression pattern of the OOMT-GFP protein in rose
petal cells was due to its being associated with a
specific membrane compartment of the cell.
OOMT-Like Genes Are Present, But Are Not Expressed,
in European Roses, Which Do Not Produce Phenolic
Methyl Ethers
The above experiments provided a description of
OOMT expression at the organ and cellular levels in
DMT-producing roses and indicated that European
species did not emit DMT because they did not express
OOMT enzyme activity. In the genus Clarkia, there
is evidence that evolution of the ability to emit
(S)-linalool involved up-regulation of an (S)-linalool
synthase gene that is also present in unscented Clarkia
species (Dudareva et al., 1996). We were therefore
interested to determine whether the European rose
species possessed OOMT gene sequences in their
genomes. PCR amplifications were carried out using
genomic DNA from a range of rose species and varieties and oligonucleotides based on Old Blush OOMT
sequences. In addition to R. gallica and Damask rose,
we also selected the wild Chinese species R. gigantea,
which, like R. chinensis, is another progenitor of
modern roses, for this study. The flowers of R. gigantea
emit almost exclusively DMT, like the flowers of the
modern Tea rose R. x hybrida cv Lady Hillingdon,
which was also included in this study (Joichi et al.,
2005). DNA fragments of 1.3 kb could be amplified
from the DNA of all the rose varieties tested (Fig. 5).
These fragments were cloned and 10 PCR products
were sequenced for each variety. For R. chinensis cv
Old Blush, all sequenced clones corresponded either to
the OOMT1 cDNA (accession no. AJ439741; Scalliet
et al., 2002) or to the OOMT2 cDNAs (accession no.
AJ439742), and harbored a 220-bp insertion, which
possessed all the characteristics of an intron. Similarly,
two sequenced clones amplified from R. x hybrida cv
Lady Hillingdon corresponded to the previously described OOMT1-related gene, OOMT3 (accession no.
AJ439743), with minor differences probably due to
allelic variations. Two other genes amplified from
Lady Hillingdon were similar, but not identical, to
the previously described OOMT4 (accession no.
AJ439744; 97% and 97.3% sequence identity, respectively). These sequences also possessed a 220-bp intron. Four different putative OOMT genes were cloned
from R. gigantea, all very similar to Old Blush OOMT
genes. Interestingly, the genomic DNA fragments
amplified from the European species R. gallica and
Damask rose also showed high similarity to R. chinensis OOMT genes. Three different types of amplified
genomic fragments were cloned from Damask rose,
two of which were extremely similar to R. chinensis
OOMT genes (98.9% and 97.5% sequence identity with
OOMT1, respectively), with the third being more
distantly related (92.5% sequence identity). Two different types of amplified genomic fragments were
cloned from R. gallica, both very similar to R. chinensis
OOMT genes (98.9% and 97.6% sequence identity with
OOMT1, respectively). Taken together, these data
showed that European roses, such as R. gallica and
Damask rose, possess sequences that are highly similar to previously characterized OOMT genes despite
the fact that no OOMT activity could be detected in
their petals. The deduced protein sequences were very
similar to R. chinensis OOMTs (Fig. 6), and analysis
of the alignment of these putative rose OOMTs with
R. chinensis OOMTs indicated that these predicted
proteins contain all the conserved sequences necessary
for OMT activity. In phenolic methyl ether-producing
Figure 5. Presence of OOMT-like genes in European roses. Agarose gel
analysis of the products of PCR reactions using OOMT-specific primers
and genomic DNA from different rose varieties as a template. R.c.,
R. chinensis cv Old Blush; R.d., Damask rose; R.g., R. gallica; R.gig., R.
gigantea; R.h., R. x hybrida cv Lady Hillingdon; M, size marker.
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Figure 6. Alignment of proteins deduced from OOMT genes. The complete amino acid sequence of OOMT1 from Old Blush
(OB OOMT1, accession no. AJ439741) is shown, and only the variant amino acid residues from the remaining 14 sequences
are indicated. Other OOMTs and putative OOMTs are deduced from OOMT genes in Old Blush (OB OOMT2, AJ439742), R. gigantea
(RgiOOMT1, AJ786313; RgiOOMT2A, AJ786314; RgiOOMT2B, AJ786315; RgiOOMT2C, AJ786316), Lady Hillingdon (LH
OOMT3A, AJ786309; LH OOMT3B, AJ786310; LH OOMT4A, AJ786311; LH OOMT4B, AJ786312), R. gallica (RgaOOMT2A,
AJ786304; RgaOOMT2B, AJ786305), and Damask rose (RdaOOMT2A, AJ786306; RdaOOMT2B, AJ786307; RdaOOMT2C,
AJ786308). The percentage identity of the different OOMT proteins, compared to OOMT1 from Old Blush, is indicated.
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Scalliet et al.
rose varieties, OOMT genes have been shown to be
strongly expressed in petals (Lavid et al., 2002; Scalliet
et al., 2002). However, when reverse transcription
(RT)-PCR was used to screen for OOMT transcripts
in a range of floral organs (sepals, petals, stamens,
pistils) and in young leaves of R. gallica and Damask
rose, no OOMT expression could be detected in any of
the tested tissues, despite repeated tests using the
same OOMT-specific primers as were used to amplify
the genomic sequences (Fig. 7).
To determine whether the OOMT homologs from
R. gallica potentially encode functional proteins, the
OOMT2A and OOMT2B sequences from this species (see Fig. 6) were expressed in tobacco (Nicotiana
tabacum) using Agrobacterium-mediated transient
transformation (Batoko et al., 2000). As controls, the
plasmids encoding GFP fused either to the N terminus
or C terminus of OOMT1 from Old Blush (the OOMTGFP and GFP-OOMT constructs in Fig. 3, J–L) were also
transformed into tobacco leaves (Fig. 8A). Control extract, from a leaf expressing GFP alone, exhibited a background level of OOMT activity, indicating that tobacco
leaves possess OMTs, which are able to efficiently methylate orcinol to produce 3-methoxy 5-hydroxytoluene
(MHT). However, OOMTactivity was significantly higher
in leaf sectors expressing GFP-OOMT or OOMT-GFP,
indicating that the OOMT moieties of both fusion
proteins were properly folded and active. As no cDNA
was available, the OOMT2A and OOMT2B genes of
R. gallica were cloned into the pK7FWG2 vector (Karimi
et al., 2002) and transformed into tobacco. Western-blot
analysis of infiltrated leaves showed that the 200-bp intron of both OOMT2A and OOMT2B genes was properly
spliced and that the OOMT2A-GFP and OOMT2B-GFP
fusion proteins were produced as efficiently as the
OOMT-GFP control protein (Fig. 8B). The OOMT activity
in cell-free extracts of tobacco leaves sectors expressing
OOMT2A-GFP and OOMT2B-GFP fusion proteins was
higher than in the control extract (leaf sector expressing
GFP only), indicating that OOMT2A and OOMT2B from
R. gallica possess OOMT activity, although the R. gallica
enzymes did not methylate orcinol as efficiently as
OOMT1 from Old Blush (Fig. 8C).
Figure 8. Characterization of OOMT-GFP fusion proteins expressed in
tobacco. A, TLC analysis of OOMTactivity in tobacco leaves transiently
expressing GFP and GFP fusion proteins. Tobacco leaf sectors expressing GFP, or GFP fusion proteins, were excised 48 h after Agrobacterium-mediated transformation. Purified recombinant R. chinensis cv
Old Blush OOMT1 (1) and cell-free extracts (20 mg proteins) of leaf
sectors expressing GFP (2), Old Blush GFP-OOMT (3), or Old Blush
OOMT-GFP (4) were incubated with orcinol in the presence of
S-adenosyl-L-[methyl-14C]Met. Arrows indicate the positions of the
origin (Ori) and the reaction product MHT. B, Western-blot analysis of
OOMT-GFP fusion proteins transiently expressed in tobacco. Cell-free
extracts (20 mg proteins) of leaf sectors expressing GFP (1), Old Blush
OOMT-GFP (2), R. gallica OOMT2A-GFP (3), and R. gallica OOMT2BGFP (4) were subjected to SDS-PAGE and western blot using antiOOMT antibody. C, OOMT activity in cell-free extracts (20 mg proteins)
of leaf sectors expressing GFP (1), Old Blush OOMT-GFP (2), R. gallica
OOMT2A-GFP (3), and R. gallica OOMT2B-GFP (4) incubated
with orcinol in the presence of S-adenosyl-L-[methyl-14C]Met. Bars
indicate 6SEs.
Together, these experiments showed that, although
European roses such as R. gallica and Damask rose
possess OOMT homologs encoding potentially active
proteins, these genes are not expressed in flower
tissues and this would therefore explain the absence
of DMT production in these species.
Figure 7. RT-PCR analysis of OOMT gene expression in different
organs of Damask rose, R. gallica, and R. chinensis cv Old Blush.
Shown is agarose gel analysis of the RT-PCR reaction products using
OOMT-specific primers (top section) and RNA extracted from leaves
(L), sepals (Se), petals (P; stage 4), and stamens 1 pistils (St/Pi).
Glyceraldehyde-3-P dehydrogenase was used as a control (bottom
section). The positions of DNA size markers are shown to the left in
kilobases (kb).
DISCUSSION
OOMTs Accumulate Specifically in Petals and
Predominantly in the Conical Cells of the
Adaxial Epidermis
In most roses, petals are the major site of scent biosynthesis, although in some cases volatile compounds
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Biosynthesis of Scent in Rose Petals
are also produced in stamen and sepals (Dobson et al.,
1990). In previous studies, transcripts of rose OOMT
genes were detected in petals and, to a lesser extent, in
stamens, but were not detected in other parts of the
flower or in leaves (Lavid et al., 2002; Scalliet et al.,
2002). Using a polyclonal antibody raised against
recombinant OOMT1, we show here that OOMTs
accumulate specifically in petals. No OOMT protein
was detected in stamens, despite the presence of
transcripts in this organ, indicating that OOMT expression is repressed in stamens by a posttranscriptional mechanism. In petals, the accumulation of
OOMT proteins followed the previously described
pattern of transcript accumulation, and enzyme levels
reached a maximum in the freshly open flower (stage
4), when scent biosynthesis is most active (Lavid et al.,
2002).
The adaxial epidermis of rose petals is composed of
specialized, cone-shaped secretory cells, and it has
been suggested, based on cytological and biochemical
analyses, that the epidermis functions as a layer of
secretory cells responsible for the biosynthesis and
emission of scent compounds (Stubbs and Francis,
1971; Loomis and Croteau, 1973). Immunolocalization
experiments (Fig. 3, A–C) showed that the majority of
the OOMT protein was concentrated in the adaxial
epidermis in petals of R. x hybrida cv Lady Hillingdon,
providing strong support for this hypothesis. A lower
level of OOMT protein also accumulated in the abaxial
epidermis, indicating a probable differential role for
the two petal epidermal layers, the adaxial epidermis
being the major site for scent production. A similar
localization pattern has been described for another
enzyme involved in flower scent biosynthesis,
S-adenosyl-L-Met:benzoic acid carboxyl methyltransferase. S-adenosyl-L-Met:benzoic acid carboxyl methyltransferase protein was detected predominantly in
the conical cells of the inner epidermal layer and, to a
lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes, providing strong support
for the commonly held belief that petal scent biosynthesis occurs in the epidermis (Kolosova et al.,
2001).
Taken together with previous studies, these data
indicate that the cone-shaped cells covering the petals
of many flowers play a major role in attracting pollinators by using both visual and olfactory cues. From
a visual point of view, the conical shape of the epidermal cells enhances light absorption by the floral
pigments and, thus, the intensity of their color (Gorton
and Vogelmann, 1996). This study and that of Kolosova
et al. (2001) indicate that the cone-shaped epidermal
cells also play a major role in the production of olfactory
cues, harboring high concentrations of enzymes involved in scent biosynthesis pathways.
Petal Epidermal Cells as a Secretory Cell Model
To date, little is known about the intracellular biosynthesis of volatile compounds in secretory cells and
the trafficking of these compounds from their sites of
synthesis to their site(s) of emission (Kolosova et al.,
2001). Peppermint glandular trichomes have been successfully used as secretory cell model system to investigate the metabolism of monoterpenes (Mahmoud
and Croteau, 2002), and peppermint trichomes have
been well characterized at the structural level (Turner
et al., 2000). The immunolocalization of several monoterpene biosynthetic enzymes (Turner and Croteau,
2004) showed that monoterpene biosynthesis may
involve the trafficking of intermediate products between different cell compartments; however, the cellular events leading to the biosynthesis and secretion
of monoterpenes remain poorly understood. In recent
years, the use of GFP as an in vivo reporter has
emerged as an extremely valuable tool in plant cell
biology (Hawes et al., 2001). However, due to the lack
of a suitable model system, plant secretory cells have
not been investigated using this technology. Rose petal
epidermal cells present a distinct advantage for this
type of approach, compared with the dispersed glandular trichomes from peppermint, for example, because they constitute a homogenous layer, covering
the whole surface of the petal and are therefore
particularly well suited for biolistic delivery of gene
constructs. Moreover, characterization of the internal
structure of the rose petal secretory cell, using GFPfusion proteins targeted to different cellular compartments, indicated a similar general structure to more
commonly studied cellular models such as tobacco or
onion (Allium cepa) epidermis cells (Boevink et al.,
1998; Scott et al., 1999; Brandizzi et al., 2004), providing
the possibility of comparative studies of secretory and
nonsecretory cell types. Biolistic transformation can be
applied to the petals from other plant species (Clark
and Sims, 1994; Quattrocchio et al., 1999), and the
results presented in this study therefore underline the
potential of petal epidermal cells as a secretory cell
model system.
Association of Rose Petal OOMTs with Cell Membranes
There is considerable evidence supporting a role for
the ER in the biosynthesis of secondary metabolites.
For example, immunolocalization experiments have
shown that cytochrome P450 limonene 6-hydroxylases
was bound to smooth ER in spearmint (Mentha spicata;
Turner and Croteau, 2004). In alfalfa (Medicago sativa),
biosynthesis of the phytoalexin medicarpin involves
the hydroxylation of liquiritigenin by the ER-associated
cytochrome P450 isoflavone synthase and the subsequent methylation of one hydroxyl group by isoflavone O-methyltransferase (IOMT). After elicitation of
the isoflavonoid pathway, IOMT localizes to the ER,
where it probably interacts with isoflavone synthase to
ensure efficient metabolic channeling and hence optimize production of medicarpin (Liu and Dixon, 2001).
In rose petals, both biochemical fractionation and
confocal microscopy studies indicated that a significant fraction of the OOMT protein was strongly bound
Plant Physiol. Vol. 140, 2006
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Scalliet et al.
to membranes throughout organ development, with
the proportion of membrane-bound OOMT increasing
as the petal matured (Fig. 4). When an OOMT-GFP
fusion protein was expressed in these cells, fluorescence was associated with compact structures, which
were often, but not always, localized at the apical pole
of the cells. In contrast to the situation observed for
alfalfa IOMT after elicitation (Liu and Dixon, 2001),
the fluorescence pattern observed after expression of
OOMT-GFP fusion protein was very different from the
pattern obtained with ER-targeted GFP-HDEL (Fig.
3I), indicating that the OOMTs were not associated
with the ER. The localization of OOMT-GFP to compact cellular structures appears to involve the N
terminus of the protein, as the N-terminal GFPOOMT fusion protein was not concentrated in the
same manner but was dispersed freely in the cytosol.
Expression of GFP-OOMT and OOMT-GFP in tobacco,
using Agrobacterium-mediated transient expression,
showed that both fusion proteins retained OOMT
activity (Fig. 8A), indicating that their OOMT moieties
were properly folded and suggesting that the fluorescence pattern observed in rose petals did not correspond to artefactual aggregates, but rather to a
physiologically relevant localization of the OOMT
protein. It is unlikely that the OOMTs were targeted
to organelles such as vacuoles because they lack any
predicted signal peptide, according to programs such
as TargetP (Emmanuelsson et al., 2000). Furthermore,
membrane-bound and soluble forms of OOMTs exhibited the same electrophoretic mobility, so it is
unlikely that they possess a cleaved signal peptide.
This also suggests that it is unlikely that membrane
association involves the attachment of a lipid moiety
to the protein as observed for chalcone isomerase
(Burbulis and Winkel-Shirley, 1999). One possibility is
that OOMTs were bound to the cytosolic side of the
membranes of vacuolar compartments. Indeed, the
structures revealed by OOMT-GFP fluorescence could
be related to storage vacuoles, which have been
shown to be distinct from the central lytic vacuole
(Paris et al., 1996). Further subcellular markers (Di
Sansebastiano et al., 1998) will be used to address this
question.
It is intriguing that the proportion of membraneassociated OOMT increased during rose petal maturation, and this may represent a mechanism for the
regulation of enzyme activity. For this reason, we
were interested in determining whether OOMT1 and
OOMT2 showed the same pattern of membrane association during development. Anti-OOMT antibodies
recognized both OOMT1 and OOMT2 and did not
allow discrimination between these two proteins.
However, biochemical analysis, using the different
substrate specificities of OOMT1 and OOMT2 to distinguish between them (Lavid et al., 2002; Scalliet et al.,
2002), indicated that both soluble and membranebound OOMT fractions exhibited the same intermediate substrate specificity, suggesting that neither
OOMT1 nor OOMT2 associated preferentially with
membranes (data not shown). Future work will be
aimed at investigating the physiological significance of
the association of OOMTs with these membrane structures. In particular, it will be important to identify the
cell structures that OOMTs are bound to, for example,
using immunogold staining and electron microscopy
techniques, such as those used successfully for other
scent-related enzymes (Kolosova et al., 2001).
Why Don’t All Roses Produce DMT?
Most modern rose varieties produce DMT as a component of their scent, having inherited this trait from
their Chinese progenitors (Nakamura, 1987; Flament
et al., 1993; Joichi et al., 2005). However, while DMT
production is almost universal in modern rose varieties, this is not true for older European varieties, which
do not produce phenolic methyl ethers (Flament et al.,
1993). Investigating the reasons for this absence in two
European roses, R. gallica and Damask rose, we found
that neither OOMT activity nor OOMT protein could
be detected in the floral organs of these varieties. The
genomes of both R. gallica and Damask rose contain
OOMT homologous sequences, but these genes are not
expressed at detectable levels in either floral organs or
leaves. There are many other examples of related plant
species exhibiting very different scent characteristics
(Dudareva et al., 1996; Gang et al., 2002; Guterman
et al., 2002), and in some cases this has also been
shown to be a result of changes in gene expression. For
example, the scented C. breweri is believed to have
evolved recently from the nonscented Clarkia concinna
(Dudareva et al., 1996). In C. breweri, the gene encoding
linalool synthase (Lis) is highly expressed in stigma
and in the epidermal cells of petals, as well as in
stamens, whereas in the nonscented C. concinna, Lis is
expressed only in the stigma and at a relatively low
level (Dudareva et al., 1996). Thus, it appears that C.
breweri has evolved its ability to emit large amounts of
S-linalool simply by increasing the expression level of
Lis in the epidermal cells of the petals.
An interesting question is the following: Did the
common ancestor of the European and Chinese rose
species produce phenolic methyl ethers, with this
ability being subsequently lost by European species,
or did Chinese roses acquire the ability to carry out
this metabolism after their separation from European
species? Phylogenetic analysis of the genus Rosa shows
that the Chinese roses, grouped under the Indicae
section, may have evolved recently within this genus
(Wissemann and Ritz, 2005), supporting the idea that
new functions may have evolved in this group. For
example, Chinese roses may have evolved their ability
to produce phenolic methyl ethers as a result of a
combination of increased OOMT gene expression in
petals and evolution of more catalytically active enzymes. In this hypothesis, the up-regulation of OOMT
gene expression may have been a critical step in the
evolution of scent production in roses; however, a more
detailed analysis of the OOMT gene family will be
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Biosynthesis of Scent in Rose Petals
needed to definitively address the question of the
origin of the biosynthesis of phenolic methyl ethers in
the genus Rosa.
MATERIALS AND METHODS
Plant Materials
Two main types of roses were used for this study: on one hand, the Chinese
roses Rosa chinensis cv Old Blush and Rosa gigantea, and the modern varieties
Rosa x hybrida cv Lady Hillingdon and R. x hybrida cv Anna (PEKcougel),
which produce phenolic methyl ethers; and on the other hand, the European
species Rosa gallica officinalis and Damask rose (Rosa damascena), which do not
produce these scent compounds. Old Blush, R. gallica officinalis, and Damask
rose were grown in open soil conditions at the Ecole Normale Supérieure de
Lyon. R. gigantea and Lady Hillingdon were from the Lyon Botanical Garden.
Anna flowers were obtained from the local producer Hortirose and grown
under greenhouse conditions. Flower development was divided into six
stages, according to Guterman et al. (2002). At stage 1, flower buds are closed.
At stage 2, petals start to emerge from the sepals. Stages 3 and 4 are characterized by petal elongation. At stage 5 and 6, petals unroll to reach full size.
Chemicals and Radiochemicals
MHT was prepared according to Scalliet et al. (2002). S-adenosyl-L[methyl-14C]Met (55 mCi/mmol) was from Amersham. All other chemicals
and reagents were from Sigma.
Preparation of Cell-Free Extracts and Microsomes
Rose petals or tobacco (Nicotiana tabacum) leaves were homogenized in
buffer A (0.1 M Tris, pH 7.5, containing 20% glycerol [v/v], 5 mM MgCl2, 10 mM
NaF, 14 mM 2-mercaptoethanol, and 1% phenylmethylsulfonyl fluoride) with
1% (w/w) polyvinylpolypyrrolidone, using 4 mL of buffer A per gram fresh
weight. The homogenate was filtered through glass wool and centrifuged at
5,000g for 15 min at 4°C. The supernatant was used for enzyme assays. For
microsome preparation, the same 5,000g supernatant was centrifuged at
150,000g for 1 h at 4°C. The resultant pellet was washed twice with buffer A,
and then resuspended in the same buffer.
Measurement of Enzyme Activity
Cell-free rose petal or tobacco leaf extracts were incubated in a final
volume of 50 mL with 50 mM S-adenosyl-L-[methyl-14C]Met and 1 mM of
orcinol in buffer A, and the incorporated radioactivity was measured by liquid
scintillation. Reaction products were analyzed by thin-layer chromatography
(TLC) on silica gel (Merck) with chloroform as the solvent, using a Storm 860
phosphoimager (Molecular Dynamics). Enzyme reaction products were
identified by comigrating with standards.
Protein Purification and Production of Antibodies
OOMT1 coding regions were cloned into pGEX-4T1. Recombinant OOMT1
protein was expressed, purified, and recovered by cleavage with thrombin as
by Scalliet et al. (2002). Polyclonal antibodies, generated in rabbits against the
purified recombinant OOMT1, were prepared by Eurogentec.
SDS-PAGE and Western-Blot Analysis
Based on the petal homogenate volume, equivalent amounts of soluble and
microsomal proteins were resolved on 10% Tris-Gly gels using a Mini Protean
II gel apparatus (Bio-Rad) and transferred to nitrocellulose membranes using
a Bio-Rad Trans-Blot apparatus. The membranes were blocked with 2% (w/v)
bovine serum albumin (BSA) in Tris-buffered saline (TBS) and probed with
a primary antibody against recombinant OOMT1 at a dilution of 1:1,000 in 2%
(w/v) BSA in TBS containing 1% (v/v) Tween 20. Goat anti-rabbit IgG
horseradish peroxidase conjugate was used as the secondary antibody at
a dilution of 1:5,000 in TBS containing 1% (v/v) Tween 20, with visualization
using a chemiluminescence assay kit (ECL; Amersham Biosciences).
Plant Vectors
For expression of the N-terminal GFP fusion protein (GFP-OOMT), OOMT
coding sequences were amplified by PCR using the upstream primer
5#-AAAAAGCAGGCTATGGAAAGGCTAAACAGCTTTAGACA-3# and the
downstream primer 5#-AGAAAGCTGGGTCAGGATAAACCTCAATGAGAGACCTTAA-3#. For expression of the C-terminal GFP fusion protein (OOMTGFP), OOMT coding sequences were amplified by PCR using the upstream
primer 5#-AAAAAGCAGGCTCCATGGAAAGGCTAAACAGCTTTAGACA-3#
and the downstream primer 5#-AGAAAGCTGGGTTCAAGGATAAACCTCAATGAGAGACC-3#. Amplified DNA fragments were introduced into
pDONR 201 (Invitrogen) and then into pK7WGF2 (GFP-OOMT) and
pK7FWG2 (OOMT-GFP; Karimi et al., 2002) using GATEWAY cloning technology (Invitrogen), according to the supplier’s instructions. Alternatively, for
a higher expression efficiency of the OOMT-GFP fusion protein in rose petals,
OOMT1 coding sequence was cloned into the NcoI site of the vector pCATSGFP, which contains a codon-optimized GFP S65C mutant gene (Heim et al.,
1995) under the control of the cauliflower mosaic virus 35S promoter and an
additional translation enhancing element of Tobacco etch virus. OOMT1 coding
sequence was amplified by PCR using the upstream primer 5#-GCGTCATGATAAGGCTAAACAGCTTTAAACACCTTAAC-3# and the downstream
primer 5#-CGCTCATGATAGGATAAACCTCAATGAGAGACCTTAAA-3#; the
amplified DNA fragment was cleaved with BspHI and cloned into the NcoI
site of the pCATS-GFP vector.
Transient Expression of GFP Fusions
For biolistic transformation, plasmid DNA (5 mg) was mixed with 50 mL of
an aqueous suspension containing 7.5 mg of 1.0 mm gold particles (Bio-Rad).
The gold DNA suspension was dispersed by vortexing in the presence of 1.25 M
CaCl2 and 17 mM spermidine and kept on ice for 10 min. The DNA-coated
gold particles then were collected by brief centrifugation, washed, resuspended in ethanol, and spread onto carrier discs for biolistic bombardment using
the particle delivery system 1000/He (Bio-Rad). Young (stage 3) rose petals
were excised and placed on moist filter paper in petri dishes. The gold
particles were fired at 1,100 p.s.i., and bombarded petals were maintained in
the dark at 22°C before examination. For Agrobacterium-mediated transient
expression, each expression vector was introduced into Agrobacterium tumefaciens strain C58 (pMP90) by electroporation. Tobacco SR1 (cv Petit Havana)
leaves were infiltrated with A. tumefaciens cultures (OD600 0.1) according to
Batoko et al. (2000). Disks were punched from tobacco leaves 48 h after
Agrobacterium infiltration and cell-free extracts were assayed for OOMT
activity as described above.
Microscopy Techniques
Rose petals were examined 12 to 48 h after biolistic transformation using
a Zeiss LSM 510 confocal imaging system attached to a Zeiss Axioplan 2
microscope (Carl Zeiss). GFP was visualized with a 3 40 water immersion
apochromat objective (Zeiss) by excitation with the 488 line of a krypton/
argon laser and use of a BP 505-550 emission filter. Serial optical sections were
obtained at 1-mm intervals, and projections of optical sections were accomplished using LSM image-processing software (Zeiss). For electron microscopy observations, petals were fixed in 1.5% glutaraldehyde in 0.1 M sodium
cacodylate buffer (pH 7.2) and post fixed in 1% OsO4. Petals were then
embedded in Spurr’s resin. Paradermal ultrathin sections were prepared with
an RMC MT 6000 ultramicrotome (RMC). The sections were mounted on 100
mesh grids coated with formvar film, stained with 1% uranyl acetate and 0.3%
lead citrate, and observed using a Hitachi H-800 transmission electron
microscope. For immunolocalization, rose petals were fixed overnight at 4°C
in 3.7% formaldehyde, 5% acetic, 50% ethanol. Post fixation, dehydration,
clearing, and wax embedding were performed according to the protocol of
Jackson (1991). Petal cross sections (10 mm thickness) were first incubated for
1 h at room temperature in TBS containing 0.3% (v/v) Tween 20 and 2% (w/v)
BSA, followed directly by incubation either with anti-OOMT1 polyclonal
antibodies (1:300 dilution in the same buffer) or with the same dilution of
preimmune serum. Goat anti-rabbit IgG alkaline phosphatase conjugate was
used as the secondary antibody at a dilution of 1:1,000 in TBS containing 0.3%
Tween 20, with visualization using NBT/BCIP.
Plant Physiol. Vol. 140, 2006
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Scalliet et al.
Cloning of OOMT-Like Genes
Rose genomic DNA was prepared according to Delichère et al. (1999).
OOMT genes were amplified by PCR using the upstream primer 5#-ATGGAAAGGCTAAACAGCTTTAGACACCTTAAC-3# and the downstream
primer 5#-TCAAGGATAAACCTCAATGAGAGACCTTAAACC-3#. PCR amplification was carried out for 30 cycles of denaturation at 94°C for 30 s,
annealing at 52°C for 30 s, and extension at 72°C for 2 min with a final
extension of 5 min, in a GeneAmp PCR system 9700 cycler (Perkin Elmer).
Amplified DNA fragments were cloned into pGEM-T Easy (Promega) and the
inserts sequenced. Multiple sequence alignments were constructed using
ClustalW (Thompson et al., 1994). RT-PCR analyses of OOMT genes expression were performed as described by Channelière et al. (2002), using ExTaq
(Takara) and the OOMT-specific primers as above. Glyceraldehyde-3-P dehydrogenase (accession no. BI977235) was amplified as a control using the
upstream primer 5#-ATCCATTCATCACCACCGACTACA-3# and the downstream primer 5#-GCATCCTTACTTGGGGCAGAGA-3#.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AJ786302 to AJ786316.
ACKNOWLEDGMENTS
We would like to thank Charles Broizat (Hortirose), Alexis Lacroix,
Isabelle Desbouchages, Armand Guillermin (Ecole Normale Supérieure de
Lyon), and Christophe Ferry (Jardin Botanique de la Ville de Lyon, Parc de la
Tête d’Or) for help with plant material. We thank Mohammed Bendahmane
(Ecole Normale Supérieure de Lyon) and Guido Jach (Max-Planck Institut für
Züchtungsforschung, Cologne, Germany) for the pCATS-GFP vector. We
thank Danièle Marty-Mazars (Université de Bourgogne, Dijon), Mickaël
Michel, Fabienne Simian-Lermé (Ecole Normale Supérieure de Lyon), and
Isabelle Anselme-Bertrand (Centre de Microscopie Electronique Stéphanois)
for help with microscopy techniques. We thank Anne-Marie Thierry for
assistance with antibody preparation, Hervé Leyral and Claudia Bardoux
(Ecole Normale Supérieure de Lyon) for technical assistance, and Nadine
Paris (Centre National de la Recherche Scientifique-Unité Mixte de Recherche
6037, Université de Rouen) for helpful discussions.
Received September 2, 2005; revised November 4, 2005; accepted November 4,
2005; published December 16, 2005.
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