Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids.
ABSTRACT The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.
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ABSTRACT: Flax (Linum usitatissimum L.) is an important crop with many characteristic features such as its abundant essential ω-3 fatty acids for human nutrition. Fatty acid (FA) biosynthesis in plants, including flax, involves several consecutive steps governed by different gene families. Using in silico gene mining and comparative analysis, genome-wide gene identification and characterization were performed for six gene families related to FA biosynthesis, including KAS, SAD, FAD, KCS and FAT. We identified 91 FA-related genes from flax cv. CDC Bethune genome, from which seven previously cloned genes were validated. The newly identified 84 FA-related genes include 14 novel genes from the KAS family, two from the SAD family, 13 from the FAD2 family, three from the FAD3 family, 38 from the KCS family and 14 from the FAT family. Out of the 91 genes identified, 88 were duplicated as a consequence of recent whole genome duplication events, in which 13 FAD2 genes were hypothesized to have evolved from tandem gene duplication events followed by a whole genome duplication event and, more recently, by a single gene deletion. The six gene families described here are highly conserved in plants and have diverged anciently. These newly identified flax genes will be a useful resource for further research on FA gene cloning and expression, QTL identification, marker development and marker-assisted selection.Journal of Proteomics & Bioinformatics 10/2014; 7(10):310-326.
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ABSTRACT: Episodes of rapid speciation provide unique insights into evolutionary processes underlying species radiations and patterns of biodiversity. Here we investigated the radiation of sexually deceptive bee orchids (Ophrys).Based on a time-calibrated phylogeny and by means of ancestral character reconstruction and divergence time estimation, we estimated the tempo and mode of this radiation within a state-dependent evolutionary framework.It appears that, in the Pleistocene, the evolution of Ophrys was marked by episodes of rapid diversification coinciding with shifts to different pollinator types: from wasps to Eucera bees to Andrena and other bees. An abrupt increase in net diversification rate was detected in three clades. Among these, two phylogenetically distant lineages switched from Eucera to Andrena and other bees in a parallel fashion and at about the same time in their evolutionary history.Lack of early radiation associated with the evolution of the key innovation of sexual deception suggests that Ophrys diversification was mainly driven by subsequent ecological opportunities provided by the exploitation of novel pollinator groups, encompassing many bee species slightly differing in their sex pheromone communication systems, and by spatiotemporal fluctuations in the pollinator mosaic.New Phytologist 12/2014; · 6.55 Impact Factor
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ABSTRACT: Divergent selection by pollinators can bring about strong reproductive isolation via changes at few genes of large effect. This has recently been demonstrated in sexually deceptive orchids, where studies (1) quantified the strength of reproductive isolation in the field; (2) identified genes that appear to be causal for reproductive isolation; and (3) demonstrated selection by analysis of natural variation in gene sequence and expression. In a group of closely related Ophrys orchids, specific floral scent components, namely n-alkenes, are the key floral traits that control specific pollinator attraction by chemical mimicry of insect sex pheromones. The genetic basis of species-specific differences in alkene production mainly lies in two biosynthetic genes encoding stearoyl–acyl carrier protein desaturases (SAD) that are associated with floral scent variation and reproductive isolation between closely related species, and evolve under pollinator-mediated selection. However, the implications of this genetic architecture of key floral traits on the evolutionary processes of pollinator adaptation and speciation in this plant group remain unclear. Here, we expand on these recent findings to model scenarios of adaptive evolutionary change at SAD2 and SAD5, their effects on plant fitness (i.e., offspring number), and the dynamics of speciation. Our model suggests that the two-locus architecture of reproductive isolation allows for rapid sympatric speciation by pollinator shift; however, the likelihood of such pollinator-mediated speciation is asymmetric between the two orchid species O. sphegodes and O. exaltata due to different fitness effects of their predominant SAD2 and SAD5 alleles. Our study not only provides insight into pollinator adaptation and speciation mechanisms of sexually deceptive orchids but also demonstrates the power of applying a modeling approach to the study of pollinator-driven ecological speciation.Ecology and Evolution 12/2014; · 1.66 Impact Factor
Stearoyl-acyl carrier protein desaturases are
associated with floral isolation in sexually
Philipp M. Schlütera,b,1, Shuqing Xua,b,c, Valeria Gagliardinib, Edward Whittled, John Shanklind, Ueli Grossniklausb,
and Florian P. Schiestla
Institutes ofaSystematic Botany andbPlant Biology, University of Zürich and Zürich-Basel Plant Science Center, CH-8008 Zurich, Switzerland;cInstitute of
Integrative Biology, Swiss Federal Institute of Technology Zürich and Zürich-Basel Plant Science Center, CH-8092 Zurich, Switzerland; anddDepartment
of Biology, Brookhaven National Laboratory, Upton, NY 11973
Edited* by Wendell L. Roelofs, Cornell University, Geneva, NY, and approved February 23, 2011 (received for review September 6, 2010)
The orchids Ophrys sphegodes and O. exaltata are reproductively
isolated from each other by the attraction of two different, highly
specific pollinator species. For pollinator attraction, flowers chemi-
cally mimic the pollinators’ sex pheromones, the key components
of which are alkenes with different double-bond positions. This
study identifies genes likely involved in alkene biosynthesis, encod-
ing stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs.
The expression of two isoforms, SAD1 and SAD2, is flower-specific
and broadly parallels alkene production during flower develop-
ment. SAD2 shows a significant association with alkene production,
and in vitro assays show that O. sphegodes SAD2 has activity both
as an 18:0-ACP Δ9and a 16:0-ACP Δ4desaturase. Downstream me-
tabolism of the SAD2 reaction products would give rise to alkenes
with double-bonds at position 9 or position 12, matching double-
odes. SAD1 and SAD2 show evidence of purifying selection before,
and positive or relaxed purifying selection after gene duplication.
By contributing to the production of species-specific alkene bou-
quets, SAD2 is suggested to contribute to differential pollinator
attraction and reproductive isolation among these species. Taken
together, these data are consistent with the hypothesis that SAD2
is a florally expressed barrier gene of large phenotypic effect and,
possibly, a genic target of pollinator-mediated selection.
acyl-acyl carrier protein desaturase|isolation genes|pollination|
speciation. This statement is especially true for ecological specia-
tion, in which divergent selection pressures on key traits drive the
establishment of reproductive isolation even in the absence of
geographic barriers to gene flow (1). This process fits the genic
view of speciation, in which only few loci of large effect may be
responsible for species differentiation, whereas gene flow is possi-
ble throughout the rest of the genome (2, 3). In practice, the
challenge in studying these processes is identifying the traits under
divergentselectionandtheir genetic basis (1).Inplantswithstrong
pollinator-mediated reproductive isolation (floral isolation), how-
ever, key floral traits are direct targets of selection (1, 4). By iden-
tifying the molecular mechanisms underlying these traits, genes
directly involved in reproductive isolation (so-called “barrier” or
“isolation” genes) or even speciation can be identified (3–5).
Strong floral isolation and high pollinator specificity make sex-
ually deceptive orchids an excellent system for identifying barrier
genes (4, 6). Rewardless orchids of the genus Ophrys attract male
insects by sexual mimicry, inducing mating attempts of pollinators
with flowers, whereby pollen is transferred. The key component to
this system is the chemical mimicry of the pollinator female’s
sex pheromone (7, 8), a blend of substances consisting mostly of
cuticular hydrocarbons, e.g., alkanes and alkenes. Alkenes (un-
saturated hydrocarbons) areof special importance,and a different
eproductive isolation is a central topic in the study of evolu-
proportion of alkenes was found to be the major odor difference
among two closely related Ophrys species attracting different
pollinators (9). In Ophrys, speciation by pollinator shift has been
hypothesized, and there is evidence both for pollinator-driven
(4, 6, 9, 10). In particular, specific pollinators mediate strong floral
isolation among the coflowering closely related species O. spheg-
odes and O. exaltata by effectively preventing gene flow, whereas
other reproductive barriers are largely absent (11). These species
differ mainly in the double-bond position of their major alkenes
(9), implying that the genes underlying this alkene difference may
be barrier genes (6).
Although alkanes are common components of the wax layer
covering the aerial parts of plants (12), alkenes are rare. Alkanes
are synthesized from fatty acyl-coenzyme A (CoA) intermediates
that undergo several rounds of chain elongation from the carboxyl
terminus. These fatty acid (FA) intermediates undergo reduction
to aldehydes and decarbonylation to form alkanes, mostly pro-
ducing odd-numbered alkanes from even-numbered very-long-
chain fatty acid (VLCFA) intermediates (12, 13). Alkenes are
thought to follow the same synthesis scheme, except for the in-
troduction of double-bonds in an additional desaturation step (6).
Notably, biosynthesis of the alkenes in insect sex pheromones is
likely very different from that in plants. Although insect acyl-CoA
desaturases (which introduce the double-bond into alkene pre-
cursors) were identified as putative speciation genes (3, 5), plant
acyl-acyl carrier protein (ACP) desaturases that are responsible
for the conversion of saturated to unsaturated FAs are mostly
unrelated to their animal counterparts (14). Specifically, plant
homologs of the animal integral membrane acyl-CoA desaturases
actmostly on acyl-lipid intermediates.In contrast, soluble,plastid-
of a double-bond into the precursors of alkenes in plants (6, 10).
Double-bond insertion at position Δ9of 18:0-ACP’s carbon chain
(counting from the substituted end) by a Δ9-SAD would yield
Author contributions: P.M.S., J.S., U.G., and F.P.S. designed research; P.M.S., S.X., V.G.,
E.W., J.S., and F.P.S. performed research; P.M.S., S.X., and V.G. analyzed data; and P.M.S.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The sequences reported in this paper have been deposited in the Gen-
Bank database (accession nos. FR688105–FR688110).
1To whom correspondence should be addressed: E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
†Shorthand notation for fatty acids and their derivatives is given in C:D form where C
specifies the number of carbon atoms and D the number of double-bonds; the position x
of a cis double-bond in the carbon chain is indicated by Δx
substituted end (if applicable), or by ω-x when counting from the unsubstituted end.
when counted from the
| April 5, 2011
| vol. 108
| no. 14www.pnas.org/cgi/doi/10.1073/pnas.1013313108
18:1Δ9-ACP. This product could be elongated, to e.g., 28:1Δ19-
CoA (double-bond at position Δ19= ω-9, with ω counting from
the unsubstituted end), leading to the production of 27:1Δ9
alkenes upon decarbonylation. Therefore, species differences in
alkene composition might result from changes in gene expression
and/or enzyme activity of specific SAD-encoding genes among
species, implying that such genes are candidate barrier genes in
Here, we report the isolation of SAD homologs from O. spheg-
odes and O. exaltata and discuss their potential role as barrier
genes. Specifically, we address the following questions: (i) are
there any differences among species regarding SAD gene expres-
sion or protein structure, (ii) are such differences associated with
alkene production, (iii) are SAD proteins functional desaturases,
and (iv) is there any evidence for selection on these enzymes?
Gene Cloning of Ophrys Stearoyl-ACP Desaturase Homologs. Putative
SAD-encoding transcripts were cloned by homology to Arabi-
dopsis thaliana SSI2 (SUPPRESSOR OF SA-INSENSITIVITY2;
At2g43710), the main Δ9-SAD-encoding gene of Arabidopsis.
Three putative homologs, named SAD1–SAD3 (Fig. S1A), were
identified from cDNA of Ophrys flower labella and their full
coding sequence was obtained by RACE. SAD1 was identified
only from O. sphegodes, whereas the SAD2 and SAD3 genes were
cloned from both species. SAD3 showed only silent substitutions
between the O. sphegodes and O. exaltata alleles (hereafter,
OeSAD2 differed at the amino acid level (Fig. S1A).
Evolutionary Analysis. Homologs of A. thaliana SSI2 (Table S1)
a Bayesian inference phylogeny of plant acyl-ACP desaturases
(Fig. 1 and Fig. S2A). There was only one group of monocot
desaturases, with Ophrys SAD1 and SAD2 occupying a position
separate from SAD3. This finding indicated that the gene dupli-
cation events associated with plant desaturase diversification oc-
curred after the split of monocots and eudicots. Furthermore, the
SAD1/SAD2 dichotomy is more recent than the split of proto-
SAD1/2 and SAD3. To test for the signature of selection, a maxi-
mum likelihood-based analysis of synonymous mutations (dS;
preserving the amino acid sequence) versus nonsynonymous
mutations (dN; altering the amino acid sequence) was performed.
This analysis revealed no indication of selection for SAD3.
However, significant purifying selection (P = 0.002) was found on
the SAD1/SAD2 clade before the split of SAD1 and SAD2, and
significant positive or relaxed purifying selection (all P < 0.001)
thereafter (Fig. 1, Fig. S2, and Tables S2–S4). A more conserva-
tive exact test of synonymous and nonsynonymous sites is con-
sistent with this interpretation (Table S4).
Cuticular Hydrocarbons and Gene Expression. Because high levels of
alkenes were found on flowers, but not on leaves of Ophrys (7),
the occurrence of hydrocarbons and SAD expression in different
two species differed significantly in the levels of different alkenes,
alkenes in O. exaltata (Fig. S3 B and C). Expression of SAD1 and
SAD2 (but not SAD3) differed among mature labella from thetwo
species (Fig. 2A). Together with the finding that SAD3 was
expressed in leaf tissue lacking alkenes, this suggests that SAD1
and/or SAD2 are involved in species-specific differences in alkene
production. While alkanes were found in all tissues, most alkenes
were barely detectable in leaves/bracts, sepals/petals, and labella
from the smallest buds. The relative amount of alkenes, however,
increased throughout flower development (Fig. 2B and Fig. S3 F–
but only SAD2 expression could significantly explain the presence
detectable by pollinators (Fig. 2C and Fig. S3). Although SAD3
showed a significant association with one species-specific 9-alkene
it unlikely to be a causative factor.
plastid localized (14), we checked whether a plastid transit peptide
was predicted for Ophrys SAD proteins. For SAD1 and SAD3 (but
not SAD2), the presence of a transit peptide was predicted (Table
S5). However, moderate prediction scores and N-terminal se-
quence divergence from the well-characterized Ricinus communis
plant SAD (RcSAD) indicated that care is needed when postu-
lating the subcellular localization of the Ophrys SADs. Using a
crystal structure of RcSAD as a template, structural homology
models were generated for OsSAD1, OsSAD2, OeSAD2, and
OsSAD3 (which is identical in sequence to OeSAD3). These
models were in good overall agreement, with differences among
protein backbones localized mainly to one loop region (Fig. S1B).
Geometry around the active site and substrate-binding pocket
appeared to be mostly conserved among Ricinus and Ophrys pro-
teins, and a canonical stearic acid (18:0) substrate modeled into
RcSAD fitted into Ophrys structures similarly well (Fig. S1C). The
a marked difference in isoelectric point (Table S5). Hypothetically
substrate-interacting regions were mostly similar among OsSAD2
and OeSAD2, but OsSAD1 showed some amino acid differences
Bayesian phylogeny with branch lengths from BaseML; numbers indicate
posterior probabilities (where >0.5) next to branches. Selected branches for
orchid desaturases are labeled, and the respective dN/dSratios (from CodeML
free-ratio model) are indicated in Inset. An asterisk marks branches A, B, and
C, for which dN/dSratios are significant (P < 0.01) among one- and two-ratio
models (Tables S2–S3).
Phylogenetic analysis of SAD homologs, showing monocot clade.
Schlüter et al.PNAS
| April 5, 2011
| vol. 108
| no. 14
near the aliphatic end of the substrate-binding cavity (Fig. S1E).
Overall, homology models suggested Ophrys proteins to be func-
tional desaturases, although differences among the proteins in-
dicated they might not be functionally equivalent.
SAD Functional Characterization. Protein function of putative Oph-
rys desaturases OsSAD1, OsSAD2, and OeSAD2 was investigated
in transgenic Arabidopsis and by in vitro assays of enzyme activity.
The Ophrys SAD coding sequences were heterologously expressed
in Arabidopsis under the control of the Cauliflower mosaic virus
35S RNA promoter. None of the transgenic plant lines com-
plemented the dwarf phenotype of homozygous ssi2 mutants (SI
Methods), indicating that orchid transgenes could not fully func-
tionally replace the A. thaliana desaturase SSI2. However, the
presence of the OsSAD2 transgene was significantly associated
with changes in unsaturated C18and C16FA levels in Arabidopsis
leaf lipids, suggesting that OsSAD2 has enzymatic activity in Ara-
bidopsis (Fig. S5).
To uncover thespecificreaction catalyzed by each Ophrys SAD,
recombinant proteinswere assayed fordesaturase activityinvitro,
using acyl-ACP from regiospecifically deuterated fatty acids. For
OsSAD1, no product was detectable by gas chromatography
coupled to mass spectroscopy (GC/MS), and lack of soluble
OeSAD2 expression precluded its analysis. However, desaturase
activity was observed for OsSAD2. Consistent with the lack of
complementation of Arabidopsis ssi2 mutants, in vitro OsSAD2
activity was low. This low activity may reflect a requirement for
specific ACP or ferredoxin proteins different from those present
in enzyme assays or in Arabidopsis (cf. refs. 15 and 16). OsSAD2
was active both on 18:0 and 16:0 substrates, producing 18:1Δ9and
16:1Δ4products, respectively, as confirmed by MS of fatty acid
methyl esters (FAMEs) of reaction products and their pyrrolidine
derivatives (Fig. 3). Considering fatty acid elongation from the
carboxyl end, these desaturation products would be expected to
give rise to 9-alkenes and 12-alkenes, respectively.
Reproductive isolation between O. sphegodes and O. exaltata
depends on the attraction of two different, highly specific polli-
nator species by chemical mimicry of their sex pheromones (11).
This specificity is due to the presence of alkenes with different
double-bond positions (7–9). During development, these alkenes
accumulate in the labella of Ophrys flowers. This accumulation is
surfaces, suggesting that alkene production is tissue- and stage-
specific. Among the three putative orchid desaturases, SAD3
showed a relatively constant expression without obvious species
rather than a factor linked to alkene production. By contrast,
SAD1 and SAD2 expression broadly paralleled alkene production,
and SAD2 showed a significant association with 9- and 12-alkene
levels in O. sphegodes, supporting a functional link. SAD1 and
SAD2 probably originated by gene duplication, forming a lineage
distinct from SAD3. Purifying selection before this duplication
event suggests a conserved role of the ancestral protein. The
higher rate of amino acid change after duplication may indicate
a partialrelease fromfunctionalconstraints,although,considering
18:0-ACP substrate. (B, D, and F) 7,7,8,8-2H4-16:0-ACP substrate. (A and B) GC
trace showing assay without desaturase (control; Left) and with desaturase
(Right), with retention times (minutes) indicated. Left peak, substrate; right
peak, background FA; second peak (with desaturase only), specific reaction
product. (C and D) MS fragmentation patterns of specific FAME peaks
marked by a gray arrow in A and B, showing mass ion and depicting the
molecular structure. (E and F) MS fragmentation patterns of pyrrolidine
derivatives of FAMEs in C and D. Arrows indicate ions that are diagnostic
for the double-bond positions inferred. This analysis confirms Δ9and Δ4
double-bond positions for 18:1 and 16:1 reaction products, respectively.
GC/MS analysis of OsSAD2 desaturase assay. (A, C, and E) 12,12-2H2-
control, in flower labella (Upper), and leaves (Lower). *P < 0.05 (one-way ANOVA). Error bars indicate SEM. (B) Normalized expression of SAD1–SAD3 (Upper)
and relative amounts (%) of major hydrocarbon classes (Lower) in O. sphegodes flower labella of different developmental stages (−4, smallest bud; 0, flower
at anthesis), mature sepals/petals (SP), and leaves (L). Error bars indicate SEM. (C) Correlation of normalized O. sphegodes SAD2 expression with relative
alkene amount after f(x) = arcsin x0.5transformation, for 27:1Δ9alkene (Upper; adjusted R2= 0.48, P = 2.8·10−5), and 27:1Δ12+ 29:1Δ12alkenes (Lower;
adjusted R2= 0.32, P = 0.0009), showing regression lines.
SAD expression and hydrocarbons. (A) Mean relative expression of SAD1–SAD3 in O. sphegodes (Os) and O. exaltata (Oe), normalized to G3PDH
| www.pnas.org/cgi/doi/10.1073/pnas.1013313108Schlüter et al.
that alkenes are likely under divergent selection (9), it is also
possible that selection drove the divergence of protein function.
Taken together, these results implicate Ophrys SAD2 as a desa-
the floral pseudopheromones.
OsSAD2 is a functional desaturase capable of producing
18:1Δ9(ω-9) and 16:1Δ4(ω-12) FA intermediates from which 9-
alkenes and 12-alkenes could be synthesized (Fig. 4). However,
because housekeeping desaturase activity should be ubiquitous
and not restricted to alkene-producing tissues, other proteins
must be involved to ensure that desaturation products enter the
VLCFA elongation pathway in flowers. For example, changes in
the activities of acyl-ACP thioesterase or acyl-CoA synthetase
isoforms (12, 13) would be potential candidates. Several orchid
genera related to Ophrys produce low levels of alkenes, which
might have served as a preadaptation for sexual deception in
Ophrys (17). If so, changes in the relevant proteins should be
present in both Ophrys and related genera.
Different Ophrys species produce different alkenes, and double-
bond differences will ultimately be due to desaturation reactions.
Although several different mechanisms could potentially explain
differences in desaturation among species, it appears that the
higher expression of SAD2 in O. sphegodes contributes to higher
9- and 12-alkene levels in this species. Because OeSAD2 hardly
differs from OsSAD2 around the active site and putative sub-
strate-binding pocket (Fig. S1E), it is likely that both enzymes
catalyze the same reaction. There are, however, amino acid
changes on the surface of SAD2 (Fig. S1D), so that an additional
(e.g., specific ACP or ferredoxin isoforms) (15, 16) cannot be
ruled out. Such a change may explain why only OsSAD2 affected
unsaturated FA levels in transgenic Arabidopsis. SAD1 differs
fromSAD2byboth changeson theproteinsurfaceandchangesin
than in O. exaltata (red arrows), due to expression (and possibly functional) differences. SAD2 reaction products are elongated and converted to 9- and 12-
alkenes, the levels of which are higher in O. sphegodes than in O. exaltata. The exact source of high levels of 7-alkenes in O. exaltata is unknown. Floral
alkenes are detected by pollinators, with 9- and 12-alkenes functioning as attractants to the bee Andrena nigroaenea (the pollinator of O. sphegodes).
Conversely, the bee Colletes cunicularius (the pollinator of O. exaltata) is attracted by 7-alkenes, whereas 9-alkenes reduce this attraction. Overall, different
alkene blends in the two species lead to differential pollinator attraction associated with reproductive isolation.
Model summarizing SAD2 involvement in floral isolation among O. sphegodes and O. exaltata. SAD2 activity is higher in O. sphegodes (blue arrows)
Schlüter et al.PNAS
| April 5, 2011
| vol. 108
| no. 14
the substrate is expected to bind. However, two lines of evidence
suggest that SAD1 is not a functional desaturase: First, SAD1
expression was not significantly associated with alkene pro-
duction. Second, no evidence of SAD1 activity was detected in
either in vitro assays or transgenic Arabidopsis.
The species-specific alkene differences associated with SAD2
the two specific pollinators, the solitary bees Andrena nigroaenea
(for O. sphegodes) and Colletes cunicularius (for O. exaltata),
showed that both detect 9-alkenes (C23, C25, C27, C29) and some
12-alkenes (Andrena: C27, C29; Colletes: C29) (7, 8). Moreover,
O. sphegodes alkene blends induced mating behavior in A.
(8), indicating that 9-alkenes may inhibit mating behavior in this
pollinator. Taken together, these observations suggest that the
alkenes linked to SAD2 activity are directly involved in the spec-
ificity of pollinator attraction and, thus, reproductive isolation
among the two orchid species.
Inconclusion, ourdataareconsistent withtheproposalthatthe
SAD2 desaturase underlies the phenotypic difference in 9- and
12-alkenes among O. sphegodes and O. exaltata and, thereby, con-
tributes to differential pollinator attraction and reproductive iso-
lation among these species. SAD2 therefore represents a barrier
gene of large phenotypic effect on pollinator attraction by or-
Plant Material. Plants of O. sphegodes Miller and O. exaltata Tenore subsp.
archipelagi (Gölz & Reinhard) Del Prete were grown in a greenhouse at the
Botanic Garden of the University of Zürich. For developmental stage-specific
analysis of hydrocarbons and gene expression, inflorescences were taken on
the first day of anthesis of the first flower of a given plant, flowers and buds
were dissected, and the first open flower was used as a reference point.
Gene Cloning and Expression Analysis. Total RNA was extracted from flash-
frozen orchid tissue by using TRIzol reagent (Invitrogen) according to the
manufacturer’s instructions, followed by assessment of RNA quality and
quantity by agarose gel electrophoresis and spectrophotometry using an
ND-1000 (NanoDrop Technologies). Where necessary, RNA was further puri-
and reverse-transcribed into cDNA by using RevertAid M-MuLV H−Reverse
Transcriptase (Fermentas), an anchored oligo-dT primer, and the supplier’s
protocol. Locus-specific and/or semiquantitative PCR was carried out by using
RedTaq ReadyMix (Sigma), the supplier’s protocol scaled to 10–20 μL with
cDNA from 1 ng/μL total RNA as a template. For primers and cycling con-
ditions, see Table S6 and SI Methods. Initial amplification of orchid SAD
fragments used a nested degenerate primer approach. PCR products were
cloned into pDRIVE (Qiagen), positive clones were identified, and they were
Sanger-sequenced by using BigDye 3.1 and a 3130XL Genetic Analyzer (Ap-
plied Biosystems), as recommended by the manufacturers. Full-length coding
sequence was isolated as detailed in SI Methods, deposited in GenBank (ac-
cession nos. FR688105–FR688110), and amplified essentially as before (but
reactions also containing 0.015 units per μL Pfu DNA polymerase; Promega)
with modified PCR primers (Table S6) to engineer flanking attB sequences
during PCR, as recommended by Invitrogen. AttB-site containing PCR prod-
ucts of OsSAD1, OsSAD2, and OeSAD2 were cloned into pDONR207 by BP
recombination (Invitrogen) to give pENTR207-SAD, followed by selection on
LB agar containing 10 μg/mL gentamicin, plasmid isolation, and sequence
confirmation as described before.
GC and GC/MS Analyses. Cuticular hydrocarbons were extracted by washing
internal standard. GC was carried out as described (9), except for the use of a
lower heating rate of 4 °C/min. Retention times were compared against those
of synthetic hydrocarbon standards run with the same settings. The standards
were: C19and C21–C29 n-alkanes and odd-chain (Z)-7-C21–C25, (Z)-9-C21–C29,
anAgilent 5975 GC/MS with the same oven and column settings. Discrimination
of (Z)-11/12 alkenes is not possible with these parameters. However, double-
bond positionshavepreviouslybeen determined: Both studyspecies contain 11-
and 12-alkenes, with 12-alkenes as the predominant isomer (19, 20). FAMEs
extracted from Arabidopsis lines were analyzed by GC/MS using the same set-
tings. FAMEs from desaturase assays were analyzed as in ref. 21.
Plant Expression of Desaturases and Biochemical Activity Assay. To create
2×35S:SAD expression vectors, pENTR207-SAD entry clones were recombined
with the binary plant expression vector pMDC32 (22) by LR recombination
(Invitrogen) and selected on kanamycin. Plasmids were isolated, sequenced, and
transformed into Agrobacterium tumefaciens strain LBA4404, which was, in
turn, usedto transform A. thaliana line SALK_036854 (23) by using the floral dip
dwarf phenotype (SI Methods). Transgenic Arabidopsis plants were selected on
and 25 μg/mL hygromycin. Selected independent transgenic lines in an ssi2/ssi2
background were tested for complementation: 35S:OsSAD1 (n = 2), 35S:OsSAD2
(n = 5), and 35S:OeSAD2 (n = 2). Transgene expression (Fig. S5B) and sequence
were confirmed by RT-PCR and Sanger sequencing as described above. FAMEs
were prepared by BCl3/methanol extraction (26).
Different constructs for protein expression in Escherichia coli were made
and evaluated as detailed in SI Methods. Expression clones containing N-
terminally modified orchid desaturases in the pET9d (Novagen) expression
vector were chosen for functional analysis. In these clones, amino acids 2–5
(ELHL) were deleted to remove part of the putative chloroplast transit
peptide. Proteins were purified and assayed as described (21), with minor
modifications: only 7,7,8,8-2H4-16:0-ACP and 12,12-2H2-18:0-ACP substrates
were used in assays containing 100 μg of desaturase, incubated for 2 h at
22 °C. FAMEs were suspended in 50 μL of hexane for GC/MS analysis.
Bioinformatic and Statistical Analyses. Molecular mass and isoelectric point of
proteins were predicted by using the ExPASy Server (27) and the presence of
a chloroplast transit peptide predicted using the ChloroP 1.1 server (28).
Homology modeling was performed by using the SWISS-MODEL server (29)
and the 2.4-Å crystal structure 1OQ4 (chain A) (30) of RcSAD as a template.
Validation and quality checking of the models were done by using the
ProSA-web server (31) and Procheck software (32).
Homologs of the Arabidopsis SSI2 desaturase gene (Table S1) were
extracted from public sequence databases as detailed in SI Methods and
aligned based on amino acid sequence by using PRANK 0.91 (33). Poorly
alignable regions were excluded from downstream analysis. The GTR+I+Γ
phylogenetic analysis conducted in MrBayes 3.1.2 (35), discarding results be-
fore apparent convergence of analysis chains (burn-in 1 million of 30 million
generations). Branch lengths of the resulting consensus tree were optimized
with BaseML and used as input for CodeML, both part of the PAML 4.3 (36)
package. Different models of sequence evolution were calculated with
CodeML and compared by likelihood ratio testing. Fisher’s exact tests were
done on (non)synonymous site counts (37) by using CodeML output. Statis-
tical analyses were performed in Microsoft Excel and R 2.11.0 (38).
ACKNOWLEDGMENTS. We thank A. Bolaños, A. Boyko, S. Cozzolino,
M. Curtis, S. Kessler, M. and S. Schauer, and H. Zheng for providing labora-
tory materials, help, or source code, and M. Anisimova and M. and S. Schauer
for discussions and comments. This work was supported by Austrian Science
Fund Fellowship J2678-B16 (to P.M.S.), Swiss Federal Institute of Technology
Zürich Grant TH 02 06-2 (to F.P.S.), the University of Zürich (U.G. and F.P.S.),
and the Office of Basic Energy Sciences of the US Department of Energy (J.S.
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| April 5, 2011
| vol. 108
| no. 14