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Eugenol synthase genes in floral scent variation in Gymnadenia species

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Abstract

Floral signaling, especially through floral scent, is often highly complex, and little is known about the molecular mechanisms and evolutionary causes of this complexity. In this study, we focused on the evolution of “floral scent genes” and the associated changes in their functions in three closely related orchid species of the genus Gymnadenia. We developed a benchmark repertoire of 2,571 expressed sequence tags (ESTs) in Gymnadenia odoratissima. For the functional characterization and evolutionary analysis, we focused on eugenol synthase, as eugenol is a widespread and important scent compound. We obtained complete coding complementary DNAs (cDNAs) of two copies of putative eugenol synthase genes in each of the three species. The proteins encoded by these cDNAs were characterized by expression and testing for activity in Escherichia coli. While G. odoratissima and Gymnadenia conopsea enzymes were found to catalyze the formation of eugenol only, the Gymnadenia densiflora proteins synthesize eugenol, as well as a smaller amount of isoeugenol. Finally, we showed that the eugenol and isoeugenol producing gene copies of G. densiflora are evolutionarily derived from the ancestral genes of the other species producing only eugenol. The evolutionary switch from production of one to two compounds evolved under relaxed purifying selection. In conclusion, our study shows the molecular bases of eugenol and isoeugenol production and suggests that an evolutionary transition in a single gene can lead to an increased complexity in floral scent emitted by plants.

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Acknowledgments

The authors appreciate Prof. Natalia Dudareva and  Dr. Phillip SanMiguel for their help in sequencing cDNA libraries. Thanks are also due to Edward Connor for providing help in volatile collections and Philipp Schlüter for assisting in PAML program. We also thank the “Amt für Natur und Umwelt, Chur” for issuing collection permits. This work was financed by the Swiss National Science Foundation grant to FPS (SNF; project no. 31003A-112342) and ETH Zürich doctoral scholarship to AKG.

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  1. Alok K. Gupta, Ines Schauvinhold & Eran Pichersky

    Present address: Department of Ecology and Evolution, University of Lausanne, Le Biophore, 1015, Lausanne, Switzerland

Authors and Affiliations

  1. Institute of Systematic Botany, University of Zurich, 8008, Zurich, Switzerland

    Alok K. Gupta & Florian P. Schiestl

  2. Institute of Integrative Biology, Plant Ecological Genetics, ETH Zurich, 8092, Zurich, Switzerland

    Alok K. Gupta

  3. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Ines Schauvinhold & Eran Pichersky

  4. Laboratorio de Cinética y Fotoquímica, Centro de Investigaciones y Transferencia de Sgo. del Estero, Universidad Nacional de Santiago del Estero, Santiago del Estero, Argentina

    Ines Schauvinhold

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  1. Alok K. Gupta
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Correspondence to Alok K. Gupta.

Electronic supplementary material

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Table S1

Primers used for Gymnadenia species (DOC 35 kb)

Table S2

List of most abundant transcripts from standard cDNA library and their gene annotations based on BlastX best hits (Cutoff ≤ e-6) (DOC 37 kb)

Table S3

List of most abundant transcripts from subtraction cDNA library and their gene annotations based on BlastX best hits (Cutoff ≤ e-6) (DOC 58 kb)

Table S4

Normalized instrument response to eugenol, isoeugenol, and indole (DOC 32 kb)

Table S5

Sequences used for evolutionary analysis (DOC 73 kb)

Table S6

Analysis of selection by employing PAML software (DOC 40 kb)

Figure S1

Gene Ontology classification showing relative differential expression of transcripts between Gymnadenia odoratissima standard (Std) and subtraction (SSH) EST libraries. (DOC 149 kb)

Figure S2

Alignment of deduced amino acid sequences of Gymnadenia and other functionally characterized NADPH-dependent family members. White letters on black background without asterisk represent amino acids similarities of at least seven sequences and additional asterisks below black backgrounds illustrate conserved amino acids in all sequences. The putative NADPH-binding domain is underlined in red. Species details and Genebank accession numbers of other characterized enzymes are shown in brackets: CbEGS1 (Clarkia breweri, EF467239.1), ObEGS1 (Ocimum basilicum, DQ372812.1), PhEGS1 (Petunia hybrida, EF467241.1), CbEGS2 (Clarkia breweri, EF467240.1), PhIGS1 (Petunia hybrida, DQ372813.1), CbIGS1 (Clarkia breweri, EF467238.1) and PaAIS1 (Pimpinella anisum, EU925388.1). (DOC 651 kb)

Figure S3

GC-MS analysis showing product formation by GoEGS1 and GoEGS2 in Gymnadenia odoratissima. (DOC 195 kb)

Figure S4

RT-PCR amplification showing eugenol and (iso) eugenol synthase gene expression using flower and leaf tissues. M, EGS, and IEGS reperesent size standard , eugenol and (iso) eugenol synthase genes, respecetively. Flower samples used as: 1, 2, 7, and 8 from G. odoratissima, 3, 4, 9, and 10 from G. conopsea, and 5, 6, 11, and 12 from G. densiflora and leaves tissue used as: 13, 14, 19, and 20 from G. odoratissima, 15, 16, 21, and 22 from G. conopsea and 17, 18, 23, and 24 from G. densiflora (DOC 117 kb)

Figure S5

Evolutionary analysis for Gymnadenia EGS homologs. The numbers represent posterior probabilities in phylogenetic tree. (DOC 165 kb)

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Gupta, A.K., Schauvinhold, I., Pichersky, E. et al. Eugenol synthase genes in floral scent variation in Gymnadenia species. Funct Integr Genomics 14, 779–788 (2014). https://doi.org/10.1007/s10142-014-0397-9

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