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 doublebond 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.acyl-acyl carrier protein desaturase | isolation genes | pollination | speciation R eproductive isolation is a central topic in the study of evolution, its origin and maintenance being critical for the process of speciation. This statement is especially true for ecological speciation, 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 possible throughout the rest of the genome (2, 3). In practice, the challenge in studying these processes is identifying the traits under divergent selection and their genetic basis (1). In plants with strong pollinator-mediated reproductive isolation (floral isolation), however, key floral traits are direct targets of selection (1, 4). By identifying 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 sexually 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 ...

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

“…ISH on thin sections of labella from greenhouse-grown O. exaltata and O. sphegodes for members of all three SAD clades revealed epidermal expression (Figures 1 and S1). This is consistent with previous PCR-based expression-level data [15,16] but revealed no difference in spatial expression pattern between the different desaturase homologs in labella. Therefore, involvement in alkene synthesis cannot be ruled out for any orchid SAD based upon their spatial expression patterns.…”

“…archipelagi (Gö lz & H.R. Reinhard) Del Prete (hereafter, O. exaltata) [16]. This hypothesis was tested by RNA in situ hybridization (ISH) on sections of flower labellum, the modified petal with which pollinators interact.…”

“…SADs Are Expressed in Flower Labellum Epidermis Chemical mimicry of sex pheromones by Ophrys orchids is mediated by cuticular hydrocarbons that are likely derived from very-long-chain fatty acid (FA) biosynthesis and deposited on the flower surface as wax layer components [16][17][18]. The flower epidermis is therefore expected to express the genes controlling pollinator specificity via alkene double-bond position: SAD2 for (Z)-9-and (Z)-12-alkenes common in O. sphegodes Miller [16] and potentially other members of the three known clades of orchid SADs (SAD1-SAD6; SAD2-clade: SAD1/2; SAD3-clade: only SAD3; SAD5-clade: SAD4/5/6) [15], some postulated to be responsible for (Z)-7-alkenes abundant in O. exaltata subsp.…”

“…The flower epidermis is therefore expected to express the genes controlling pollinator specificity via alkene double-bond position: SAD2 for (Z)-9-and (Z)-12-alkenes common in O. sphegodes Miller [16] and potentially other members of the three known clades of orchid SADs (SAD1-SAD6; SAD2-clade: SAD1/2; SAD3-clade: only SAD3; SAD5-clade: SAD4/5/6) [15], some postulated to be responsible for (Z)-7-alkenes abundant in O. exaltata subsp. archipelagi (Gö lz & H.R.…”

“…The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11][12][13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O. exaltata and closely related species, such as O. sphegodes [13][14][15][16]. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5.…”