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• W2557250977 abstract "•Structural genes BSMT and BPBT cause gain of scent during bee-to-moth pollination•CNL was inactivated and severely degraded during moth-to-hummingbird pollination•CNL is a target of evolutionary change across angiosperms•Identifying functional mutations can help in the ordering of speciation events The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the loss of scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early in the transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change. The interactions of plants with their pollinators are thought to be a driving force in the evolution of angiosperms. Adaptation to a new pollinator involves coordinated changes in multiple floral traits controlled by multiple genes. Surprisingly, such complex genetic shifts have happened numerous times during evolution. Here we report on the genetic basis of the changes in one such trait, floral scent emission, in the genus Petunia (Solanaceae). The increase in the quantity and complexity of the volatiles during the shift from bee to hawkmoth pollination was due to de novo expression of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT) together with moderately increased transcript levels for most enzymes of the phenylpropanoid/benzenoid pathway. Loss of cinnamate-CoA ligase (CNL) function as well as a reduction in the expression of the MYB transcription factor ODO1 explain the loss of scent during the transition from moth to hummingbird pollination. The CNL gene in the hummingbird-adapted species is inactive due to a stop codon, but also appears to have undergone further degradation over time. Therefore, we propose that loss of scent happened relatively early in the transition toward hummingbird pollination, and probably preceded the loss of UV-absorbing flavonols. The discovery that CNL is also involved in the loss of scent during the transition from outcrossing to selfing in Capsella (Brassicaceae) (see the accompanying paper) raises interesting questions about the possible causes of deep evolutionary conservation of the targets of evolutionary change. Plants adapt to changes in pollinator availability by the evolution of new combinations of floral traits that can lead to reproductive isolation and ultimately speciation. Shifts in floral pollination syndromes have happened repeatedly in many taxa and are thought to be responsible for the rapid diversification of the angiosperms [1Mitchell R.J. Irwin R.E. Flanagan R.J. Karron J.D. Ecology and evolution of plant-pollinator interactions.Ann. Bot. (Lond.). 2009; 103: 1355-1363Crossref PubMed Scopus (135) Google Scholar, 2Grant V. Pollination systems as isolating mechanisms in angiosperms.Evolution. 1949; 3: 82-97Crossref PubMed Google Scholar, 3Fenster C.B. Armbruster W.S. Wilson P. Dudash M.R. 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Kuhlemeier C. Single gene-mediated shift in pollinator attraction in Petunia.Plant Cell. 2007; 19: 779-790Crossref PubMed Scopus (265) Google Scholar], suggesting that the number of genes available for modification of floral traits is limited. Floral scent is another important signal between plants and pollinators [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Kessler D. Kallenbach M. Diezel C. Rothe E. Murdock M. Baldwin I.T. How scent and nectar influence floral antagonists and mutualists.eLife. 2015; 4: e07641Crossref Scopus (52) Google Scholar, 23Dötterl S. Jürgens A. Seifert K. Laube T. Weissbecker B. Schütz S. 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So far, only a few genes have been associated with scent differences affecting pollinator attraction [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 12Byers K.J.R.P. Vela J.P. Peng F. Riffell J.A. Bradshaw Jr., H.D. Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus).Plant J. 2014; 80: 1031-1042Crossref PubMed Scopus (62) Google Scholar, 38Xu S. Schlüter P.M. Grossniklaus U. Schiestl F.P. The genetic basis of pollinator adaptation in a sexually deceptive orchid.PLoS Genet. 2012; 8: e1002889Crossref PubMed Scopus (37) Google Scholar]. Here we use the genus Petunia (Solanaceae) to investigate the genetic basis of loss and gain of scent emission during the shifts from bee to moth and from moth to hummingbird pollination. The ancestral short-tube species Petunia inflata is pollinated by bees, whereas the two closely related long-tube species Petunia axillaris and Petunia exserta are adapted to pollination by nocturnal hawkmoths and hummingbirds, respectively. The main emitted volatiles derive from phenylalanine (Figure 1), the same molecule that is also the precursor of the anthocyanin pigments [39Muhlemann J.K. Klempien A. Dudareva N. Floral volatiles: from biosynthesis to function.Plant Cell Environ. 2014; 37: 1936-1949Crossref PubMed Scopus (244) Google Scholar, 40Sheehan H. Hermann K. Kuhlemeier C. Color and scent: how single genes influence pollinator attraction.Cold Spring Harb. Symp. Quant. Biol. 2012; 77: 117-133Crossref PubMed Scopus (37) Google Scholar]. P. inflata has purple, UV-reflecting flowers and emits a single volatile, benzaldehyde. P. axillaris flowers are white and UV absorbing, and emit a rich blend of volatiles during the night [37Hoballah M.E. Stuurman J. Turlings T.C.J. Guerin P.M. Connétable S. Kuhlemeier C. The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta.Planta. 2005; 222: 141-150Crossref PubMed Scopus (133) Google Scholar]. The three most abundant volatiles emitted by P. axillaris, benzylalcohol, methylbenzoate, and benzaldehyde, were shown to dominate the scent profile emitted by different hawkmoth-pollinated species and also to trigger a strong and innate response from the hawkmoth pollinator Manduca sexta in sensorial and behavioral assays [37Hoballah M.E. Stuurman J. Turlings T.C.J. Guerin P.M. Connétable S. Kuhlemeier C. The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta.Planta. 2005; 222: 141-150Crossref PubMed Scopus (133) Google Scholar, 41Riffell J.A. Lei H. Christensen T.A. Hildebrand J.G. Characterization and coding of behaviorally significant odor mixtures.Curr. Biol. 2009; 19: 335-340Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 42Riffell J.A. Lei H. Abrell L. Hildebrand J.G. Neural basis of a pollinator’s buffet: olfactory specialization and learning in Manduca sexta.Science. 2013; 339: 200-204Crossref PubMed Scopus (102) Google Scholar]. Little is known about the genetics of scent production during the bee-to-hawkmoth transition. In contrast, the differences in scent emission between P. axillaris and P. exserta were previously investigated [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 43Hermann K. Klahre U. Moser M. Sheehan H. Mandel T. Kuhlemeier C. Tight genetic linkage of prezygotic barrier loci creates a multifunctional speciation island in Petunia.Curr. Biol. 2013; 23: 873-877Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. Two main scent QTLs affecting pollinator attraction were identified, one on chromosome II and one on chromosome VII. Introgression of the P. exserta chromosome II QTL into the P. axillaris background abolished methylbenzoate emission, whereas introgression of the chromosome VII QTL reduced it by ∼65%. Whereas the chromosome VII QTL was identified as encoding the R2R3-MYB transcription factor ODORANT1 (ODO1) [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 44Verdonk J.C. Haring M.A. van Tunen A.J. Schuurink R.C. ODORANT1 regulates fragrance biosynthesis in Petunia flowers.Plant Cell. 2005; 17: 1612-1624Crossref PubMed Scopus (256) Google Scholar], the identity of the chromosome II QTL is unknown [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. Interestingly, the chromosome II QTL resides in a genomic region of low recombination together with four additional QTLs [43Hermann K. Klahre U. Moser M. Sheehan H. Mandel T. Kuhlemeier C. Tight genetic linkage of prezygotic barrier loci creates a multifunctional speciation island in Petunia.Curr. Biol. 2013; 23: 873-877Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. The low recombination rate in this region raises interesting questions regarding the genomic architecture of pollination syndrome genes but also makes it difficult to fine-map the genes underlying these QTLs. Here we identify cinnamate-CoA ligase 1 (CNL1) as the gene on chromosome II that is responsible for the difference in scent between P. axillaris and P. exserta and show that the increase in quantity and quality of volatiles during the shift from the ancestral P. inflata to P. axillaris required activation of the genes encoding benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) and benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase (BPBT). In order to identify the gene(s) underlying the scent QTL on chromosome II, we constructed a near-isogenic line, IL3-2, which segregates for this genomic region in the genetic background of the P. axillaris parent (Figure 2A). Further backcrossing did not reveal additional breakpoints, and thus no reduction of the apparent size of the introgression. This was not unexpected, as the QTL resides in a region of suppressed recombination [43Hermann K. Klahre U. Moser M. Sheehan H. Mandel T. Kuhlemeier C. Tight genetic linkage of prezygotic barrier loci creates a multifunctional speciation island in Petunia.Curr. Biol. 2013; 23: 873-877Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. RNA sequencing (RNA-seq) data from the three IL3-2 genotypes, homozygous for P. exserta (IL3-2P.exs) and P. axillaris (IL3-2P.axi) and heterozygous (IL3-2het), were compared to the P. axillaris genome [45Bombarely A. Moser M. Amrad A. Bao M. Bapaume L. Barry C.S. Bliek M. Boersma M.R. Borghi L. Bruggmann R. et al.Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.Nat. Plants. 2016; 2: 16074Crossref PubMed Scopus (212) Google Scholar]. This indicated that the three genotypes are homozygous for P. axillaris outside the introgressed region on chromosome II. Analysis of methylbenzoate production in the progeny of IL3-2het showed that IL3-2P.axi and IL3-2het produced high levels of methylbenzoate, whereas IL3-2P.exs produced none (Figure 2B). Therefore, the chromosome II QTL behaves as a single dominant Mendelian locus. The volatiles produced by P. axillaris contain a benzene ring but differ in the length of the side chains (Figure 1) [46Verdonk J.C. Ric de Vos C.H. Verhoeven H.A. Haring M.A. van Tunen A.J. Schuurink R.C. Regulation of floral scent production in Petunia revealed by targeted metabolomics.Phytochemistry. 2003; 62: 997-1008Crossref PubMed Scopus (214) Google Scholar]. For a more complete understanding of the QTL effect, we determined the floral emission of the C6-C1, C6-C2, and C6-C3 classes of volatiles (Figure 2C). Whereas the emission of the C6-C1 compounds benzaldehyde, benzylbenzoate, and methylbenzoate was severely reduced in IL3-2P.exs compared to IL3-2P.axi, the emission of isoeugenol (C6-C3) was elevated. No measurable emission of any compound was detected in the P. exserta parent. This suggests that the chromosome II QTL has opposite effects on the emission of different branches of the phenylpropanoid/benzenoid pathway. It is possible that the QTL specifically affects the C6-C1 pathway and thereby causes metabolic flux to be redirected toward the C6-C3 pathway. Iso/eugenol emission in IL3-2P.exs displayed a rhythmic profile, peaking during the night, similar to the methylbenzoate profile seen in P. axillaris (Figure 2D). Therefore, the chromosome II QTL appears not to affect the circadian rhythm of P. axillaris, typical for plants visited by nocturnally active pollinators [37Hoballah M.E. Stuurman J. Turlings T.C.J. Guerin P.M. Connétable S. Kuhlemeier C. The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta.Planta. 2005; 222: 141-150Crossref PubMed Scopus (133) Google Scholar]. To identify candidate genes underlying the QTL, we conducted an RNA-seq experiment and focused on genes encoding 18 proteins that were reliably annotated as enzymes of the shikimate, arogenate, and phenylpropanoid/benzenoid pathways, as well as five known transcription factors (Figure 3A; Table S1). Mapping the SNP genotypes of the expressed genes to the P. axillaris genome showed that two of the three genes encoding cinnamate-CoA ligase (CNL1 and CNL3) as well as the single gene encoding transcription factor EMISSION OF BENZENOIDS II (EOBII) reside within the introgression. Because the EOBII gene has no obvious functionally relevant SNPs in its coding region and was equally expressed in all genotypes, it was not further investigated. Of the CNL genes, only CNL1 was expressed at an appreciable level in P. axillaris. Expression of CNL1 was 20-fold lower in IL3-2P.exs and essentially undetectable in P. exserta, encouraging us to focus on this gene. Before further discussing CNL1, we make the following observations. First, of the genes outside the introgression, only the two genes encoding 4-coumarate:CoA ligase (4CL) were significantly more highly expressed in IL3-2P.axi than in IL3-2P.exs (Figure 3A). These 2- to 3-fold differences in expression are statistically robust but their biological relevance may be minor, especially when considering that suppression of 4CL1 by RNAi did not affect the scent profile [47Klempien A. Kaminaga Y. Qualley A. Nagegowda D.A. Widhalm J.R. Orlova I. Shasany A.K. Taguchi G. Kish C.M. Cooper B.R. et al.Contribution of CoA ligases to benzenoid biosynthesis in Petunia flowers.Plant Cell. 2012; 24: 2015-2030Crossref PubMed Scopus (108) Google Scholar]. Second, a comparison between P. axillaris and IL3-2P.axi revealed no significant differences in expression for any of the genes, confirming that IL3-2 is highly similar to P. axillaris outside the introgression. Third, all genes examined except the transcription factors EOBII and MYB4 were expressed at least 3-fold higher in IL3-2P.exs than in P. exserta (Table S1). This indicates the involvement of regulatory genes outside the introgression. We previously identified the transcription factor ODO1 as the gene underlying a QTL on chromosome VII [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. Because ODO1 is not known to regulate genes encoding enzymes involved in the final steps of the volatile phenylpropanoid/benzenoid pathway [44Verdonk J.C. Haring M.A. van Tunen A.J. Schuurink R.C. ODORANT1 regulates fragrance biosynthesis in Petunia flowers.Plant Cell. 2005; 17: 1612-1624Crossref PubMed Scopus (256) Google Scholar], we assume the influence of an additional regulatory factor, potentially underlying a QTL on chromosome III that showed an effect on the quantity, but not the composition, of compounds [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. qPCR analysis revealed that CNL is expressed in a diurnal rhythm. Expression peaks in the middle of the light period, and thus precedes peak volatile emission by approximately 8 hr. Compared to P. axillaris, CNL transcript levels were background levels in P. exserta. Again, we attribute the residual expression in IL3-2P.exs to regulatory loci outside the introgression (Figure 3B) [9Klahre U. Gurba A. Hermann K. Saxenhofer M. Bossolini E. Guerin P.M. Kuhlemeier C. Pollinator choice in Petunia depends on two major genetic loci for floral scent production.Curr. Biol. 2011; 21: 730-739Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. Decreased expression can be caused by mutations in the gene itself (cis effects), for instance in the promoter, or by the inactivation of a regulator (trans effect), for instance a transcription factor. To discriminate between these two possibilities, we performed allele-specific expression (ASE) analysis in IL3-2het. Expression from the P. axillaris allele was 16-fold higher than from the P. exserta allele (Figure 3C), whereas the two alleles of the EOBII gene, which served as a control, were equally expressed. This indicates that the difference in CNL expression between the two species can be attributed to a defect in the P. exserta CNL gene. CNL, also known as acyl-activating enzyme, is a peroxisomal enzyme that conjugates cinnamic acid to coenzyme A, the first committed step in the C6-C1 pathway [47Klempien A. Kaminaga Y. Qualley A. Nagegowda D.A. Widhalm J.R. Orlova I. Shasany A.K. Taguchi G. Kish C.M. Cooper B.R. et al.Contribution of CoA ligases to benzenoid biosynthesis in Petunia flowers.Plant Cell. 2012; 24: 2015-2030Crossref PubMed Scopus (108) Google Scholar, 48Colquhoun T.A. Marciniak D.M. Wedde A.E. Kim J.Y. Schwieterman M.L. Levin L.A. Van Moerkercke A. Schuurink R.C. Clark D.G. A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. ‘Mitchell Diploid’ flower.J. Exp. Bot. 2012; 63: 4821-4833Crossref PubMed Scopus (41) Google Scholar]. The enzyme contains an AMP-dependent synthetase/ligase domain with a conserved AMP binding site and a peroxisomal targeting domain (Figure 4A) [47Klempien A. Kaminaga Y. Qualley A. Nagegowda D.A. Widhalm J.R. Orlova I. Shasany A.K. Taguchi G. Kish C.M. Cooper B.R. et al.Contribution of CoA ligases to benzenoid biosynthesis in Petunia flowers.Plant Cell. 2012; 24: 2015-2030Crossref PubMed Scopus (108) Google Scholar, 48Colquhoun T.A. Marciniak D.M. Wedde A.E. Kim J.Y. Schwieterman M.L. Levin L.A. Van Moerkercke A. Schuurink R.C. Clark D.G. A peroxisomally localized acyl-activating enzyme is required for volatile benzenoid formation in a Petunia×hybrida cv. ‘Mitchell Diploid’ flower.J. Exp. Bot. 2012; 63: 4821-4833Crossref PubMed Scopus (41) Google Scholar]. Of the three CNL genes present in the P. axillaris genome, only CNL1 is expressed in post-anthesis petal tissue (Table S1). The CNL2 and CNL3 genes are expressed at background levels and have premature stop codons creating proteins that are 42 and 196 amino acids shorter, respectively [45Bombarely A. Moser M. Amrad A. Bao M. Bapaume L. Barry C.S. Bliek M. Boersma M.R. Borghi L. Bruggmann R. et al.Insight into the evolution of t" @default.
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• W2557250977 title "Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation" @default.
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