Functional & Integrative Genomics

, Volume 14, Issue 4, pp 779–788 | Cite as

Eugenol synthase genes in floral scent variation in Gymnadenia species

  • Alok K. Gupta
  • Ines Schauvinhold
  • Eran Pichersky
  • Florian P. Schiestl
Original Paper

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.

Keywords

ESTs Eugenol Floral scents Gymnadenia Isoeugenol Pollination VOC 

Supplementary material

10142_2014_397_MOESM1_ESM.doc (35 kb)
Table S1Primers used for Gymnadenia species (DOC 35 kb)
10142_2014_397_MOESM2_ESM.doc (37 kb)
Table S2List of most abundant transcripts from standard cDNA library and their gene annotations based on BlastX best hits (Cutoff ≤ e-6) (DOC 37 kb)
10142_2014_397_MOESM3_ESM.doc (58 kb)
Table S3List of most abundant transcripts from subtraction cDNA library and their gene annotations based on BlastX best hits (Cutoff ≤ e-6) (DOC 58 kb)
10142_2014_397_MOESM4_ESM.doc (32 kb)
Table S4Normalized instrument response to eugenol, isoeugenol, and indole (DOC 32 kb)
10142_2014_397_MOESM5_ESM.doc (74 kb)
Table S5Sequences used for evolutionary analysis (DOC 73 kb)
10142_2014_397_MOESM6_ESM.doc (40 kb)
Table S6Analysis of selection by employing PAML software (DOC 40 kb)
10142_2014_397_MOESM7_ESM.doc (149 kb)
Figure S1Gene Ontology classification showing relative differential expression of transcripts between Gymnadenia odoratissima standard (Std) and subtraction (SSH) EST libraries. (DOC 149 kb)
10142_2014_397_MOESM8_ESM.doc (652 kb)
Figure S2Alignment 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)
10142_2014_397_MOESM9_ESM.doc (195 kb)
Figure S3GC-MS analysis showing product formation by GoEGS1 and GoEGS2 in Gymnadenia odoratissima. (DOC 195 kb)
10142_2014_397_MOESM10_ESM.doc (117 kb)
Figure S4RT-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)
10142_2014_397_MOESM11_ESM.doc (165 kb)
Figure S5Evolutionary analysis for Gymnadenia EGS homologs. The numbers represent posterior probabilities in phylogenetic tree. (DOC 165 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Alok K. Gupta
    • 1
    • 2
    • 5
  • Ines Schauvinhold
    • 3
    • 4
  • Eran Pichersky
    • 3
  • Florian P. Schiestl
    • 1
  1. 1.Institute of Systematic BotanyUniversity of ZurichZurichSwitzerland
  2. 2.Institute of Integrative Biology, Plant Ecological GeneticsETH ZurichZurichSwitzerland
  3. 3.Department of Molecular, Cellular and Developmental BiologyUniversity of MichiganAnn ArborUSA
  4. 4.Laboratorio de Cinética y Fotoquímica, Centro de Investigaciones y Transferencia de Sgo. del EsteroUniversidad Nacional de Santiago del EsteroSantiago del EsteroArgentina
  5. 5.Department of Ecology and EvolutionUniversity of LausanneLausanneSwitzerland

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