Characterization of α-pinene synthase gene in Pinus pinaster and P. pinea in vitro cultures and differential gene expression following Bursaphelenchus xylophilus inoculation

  • Helena Trindade
  • Inês Sena
  • A. Cristina Figueiredo
Original Article


Pinus pinaster and P. pinea are two important pine species in Portugal. These two pine species show different susceptibility to Bursaphelenchus xylophilus, the nematode causing pine wilt disease, as well as a diverse volatile composition. To clarify the role of terpenes in plant–nematode interactions, the α-pinene synthase gene expression was studied, using P. pinaster and P. pinea in vitro axenic shoot cultures. Identification and isolation of α-pinene synthase genes from both pine species was performed, together with functional characterization of the genes, revealing that the translated amino acid sequences between both species shared 97.3 % pairwise identity. Heterologous expression of full and truncated sequences, devoid of the 48 amino acids of the transit peptide, proved the functionality of both, with the production of α-pinene as the major final product. Relative quantification of protein activity showed a twofold increase of α-pinene production at 4 °C in comparison to assays performed at 21 and 37 °C. Both MnCl2 and KCl were required for substrate conversion. Furthermore, the variation in gene expression was studied by RT-PCR, using both axenic in vitro shoot pine cultures and co-cultures with B. xylophilus. In P. pinaster there was no difference between co-cultures and control cultures, while in P. pinea α-pinene synthase gene was upregulated in the co-cultures, with a peak of expression at 24 hpi (h post inoculation).


Pinus In vitro culture Co-cultures Nematode invasion Monoterpenes Terpene synthases 



3′-Untranslated region


5′-Untranslated region


Amino acid


Cetyl trimethyl ammonium bromide


Diethyl pyrocarbonate





FDP (also known as FPP)

Farnesyl diphosphate (also known as farnesyl pyrophosphate)


Gas chromatography


Gas chromatography–mass spectrometry

GDP (also known as GPP)

Geranyl diphosphate (also known as geranyl pyrophosphate)


Luria–Bertani medium


Open reading frame


Polymerase chain reaction


Phenylmethylsulfonyl fluoride


Pinus pinaster α-pinene synthase


Pinus pinea α-pinene synthase


Pine wilt disease


Pinewood nematode (Bursaphelenchus xylophilus)


Reverse transcription


Schenk and Hildebrandt culture medium


Solid phase micro extraction


Westvaco culture medium



The authors would like to thank Fernando Dias from BioISI for providing the TOP10 E. coli competent cells. The authors would also like to thank the reviewers for their valuable suggestions that contributed to improve the discussion of the manuscript. This study was partially funded by Fundação para a Ciência e a Tecnologia (FCT) under research contracts PEst-OE/EQB/LA0023/2011, UID/AMB/50017/2013, FEDER PT2020-Compete 2020, and PTDC/AGR CFL/117026/2010.

Supplementary material

11738_2016_2159_MOESM1_ESM.pdf (98 kb)
Supplementary material 1 (PDF 97 kb)


  1. Abbott E, Hall D, Hamberger B, Bohlmann J (2010) Laser microdissection of Conifer stem tissues: Isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca). BMC Plant Biol 10:106–122CrossRefPubMedPubMedCentralGoogle Scholar
  2. Azevedo H, Lino-Neto T, Tavares RM (2003) An improved method for high quality RNA isolation from needles of adult maritime pine trees. Plant Mol Biol Report 21:333–338CrossRefGoogle Scholar
  3. Bohlmann J, Gershenzon J, Aubourg S (2000) Biochemical, molecular, genetic and evolutionary aspects of defense-related terpenoid metabolism in Conifers. In: Romeo JT, Ibrahim R, Varin L, De Luca V (eds) Evolution of metabolic pathways, Chapter 5. Elsevier Science, Oxford, pp 109–150Google Scholar
  4. Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bohlmann J, Steele CL, Croteau R (1997) Monoterpene synthases from Grand Fir (Abies grandis) cDNA isolation, characterization, and functional expression of myrcene synthase, (2)-(4s)-limonene synthase, and (2)-(1s,5s)-pinene synthase. J Biol Chem 272:21784–21792CrossRefPubMedGoogle Scholar
  6. CELPA (Associação da indústria Papeleira) (2014) Boletim Estatístico/Indústria Papeleira PortuguesaGoogle Scholar
  7. Coke JE (1996) Basal nutrient medium for in vitro cultures of loblolly pines. USA Patent 5.534.434Google Scholar
  8. Croteau R (1987) Biosynthesis and catabolism of monoterpenoids. Chem Rev 87:929–954CrossRefGoogle Scholar
  9. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.3. Accessed 4 Dec 2015
  10. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016CrossRefPubMedGoogle Scholar
  11. Fäldt J, Martin D, Miller B, Rawat S, Bohlmann J (2003) Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase. Plant Mol Biol 51:119–133CrossRefPubMedGoogle Scholar
  12. Faria JM, Sena I, da Silva IV, Ribeiro B, Barbosa P, Ascensão L, Figueiredo AC (2015a) In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease. Planta 241:1325–1336CrossRefPubMedGoogle Scholar
  13. Faria JM, Sena I, Moiteiro C, Bennett RN, Mota M, Figueiredo AC (2015b) Nematotoxic and phytotoxic activity of Satureja montana and Ruta graveolens essential oils on Pinus pinaster shoot cultures and P. pinaster with Bursaphelenchus xylophilus in vitro co-cultures. Ind Crops Prod 77:59–65CrossRefGoogle Scholar
  14. Ferreira de Sousa P (2000) A fileira silvo-industrial do pinheiro-bravo. In: Vieira JN, Pinto MJ, Pereira R (eds) Florestas de Portugal/Forests of Portugal. Direcção Geral das Florestas, Lisboa, pp 193–197Google Scholar
  15. Figueiredo AC, Pedro LG, Barroso JG, Trindade H, Sanches J, Oliveira C, Correia M (2014) Pinus pinaster Aiton e Pinus pinea L. Agrotec 12:23–27Google Scholar
  16. Keeling CI, Bohlmann J (2006) Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. New Phytol 170:657–675CrossRefPubMedGoogle Scholar
  17. Keeling CI, Weisshaar S, Ralph SG, Jancsik S, Hamberger B, Dullat HK, Bohlmann J (2011) Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.). BMC Plant Biol 11:43–57CrossRefPubMedPubMedCentralGoogle Scholar
  18. Mendes AC (2007) A importância económico-social do pinheiro-bravo. In: Silva JS (ed) Árvores e florestas de Portugal, vol 4., Pinhais e eucaliptais. A floresta cultivadaPúblico-FLAD, Lisboa, pp 35–46Google Scholar
  19. Mendes AC, Feliciano DM (2007) A importância económico-social do pinheiro-manso. In: Silva JS (ed) Árvores e florestas de Portugal, vol 4., Pinhais e eucaliptais. A floresta cultivadaPúblico-FLAD, Lisboa, pp 121–132Google Scholar
  20. Mendes MD, Barroso JG, Oliveira MM, Trindade H (2014) Identification and characterization of a second isogene encoding γ-terpinene synthase in Thymus caespititius. J Plant Physiol 171:1017–1027CrossRefPubMedGoogle Scholar
  21. Mota MM, Vieira PC (2008) Pine wilt disease in Portugal. In: Zhao BG, Futai K, Sutherland JR, Takeuchi Y (eds) Pine wilt disease. Springer, Japan, pp 33–38CrossRefGoogle Scholar
  22. Mota L, Figueiredo AC, Pedro LG, Barroso JG, Ascensão L (2014) Volatile oils composition and bioactivity of the essential oils of Plectranthus barbatus, P. neochilus and P. ornatus grown in Portugal. Chem Biodivers 11:719–732CrossRefPubMedGoogle Scholar
  23. Naves P, Sousa E, Quartau JA (2006) Reproductive traits of Monochamus galloprovincialis (Coleoptera: Cerambycidae) under laboratory conditions. Bull Entomol Res 96:289–294CrossRefPubMedGoogle Scholar
  24. Phillips MA, Savage TJ, Croteau R (1999) Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers. Arch Biochem Biophys 372:197–204CrossRefPubMedGoogle Scholar
  25. Phillips MA, Wildung MR, Williams DC, Hyatt DC, Croteau R (2003) DNA isolation, functional expression, and characterization of (+)-α-pinene synthase and (−)-α-pinene synthase from loblolly pine (Pinus taeda): Stereocontrol in pinene biosynthesis. Arch Biochem Biophys 411:267–276CrossRefPubMedGoogle Scholar
  26. Rodrigues AM, Mendes MD, Lima AS, Barbosa PM, Ascensão LM, Barroso JG, Pedro LG, Mota MM, Figueiredo AC (2016) Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris essential oils and monoterpene hydrocarbon enantiomers changes following inoculation with the pinewood nematode Bursaphelenchus xylophilus [submitted] Google Scholar
  27. Santos CS, Pinheiro M, Silva AI, Egas C, Vasconcelos MW (2012) Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening. BMC Genom 13:599–614CrossRefGoogle Scholar
  28. Santos CS, Vasconcelos MW (2012) Identification of genes differentially expressed in Pinus pinaster and Pinus pinea after infection with the pine wood nematode. Eur J Plant Pathol 132:407–418CrossRefGoogle Scholar
  29. Savage TJ, Hatch MW, Croteau R (1994) Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase. J Biol Chem 269:4012–4020PubMedGoogle Scholar
  30. Schenk UR, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204CrossRefGoogle Scholar
  31. Song W, Staudt M, Bourgeois I, Williams J (2014) Laboratory and field measurements of enantiomeric monoterpene emissions as a function of chemotype, light and temperature. Biogeosciences 11:1435–1447CrossRefGoogle Scholar
  32. Starks CM, Back KW, Chappell J, Noel JP (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277:1815–1820CrossRefPubMedGoogle Scholar
  33. Tingey DT, Manning M, Grothaus LC, Burns WF (1980) Influence of light and temperature on monoterpene emission rates from slash pine. Plant Physiol 65:797–801CrossRefPubMedPubMedCentralGoogle Scholar
  34. Vacas de Carvalho MA (2000) O solar do pinheiro-manso de Alcácer. In: Vieira JN, Pinto MJ, Pereira R (eds) Florestas de Portugal/Forests of Portugal. Direcção Geral das Florestas, Lisboa, pp 77–83Google Scholar
  35. Webster J, Mota M (2008) Pine wilt disease: global issues, trade and economic impact. In: Mota M, Vieira P (eds) Pine wilt disease: a worldwide threat to forest ecosystems. Springer Science, Dordrecht, pp 1–3Google Scholar
  36. Williams DC, McGarvey DJ, Katahira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active “pseudomature” form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37:12213–12220CrossRefPubMedGoogle Scholar
  37. Wise ML, Croteau R (1999) Monoterpene biosynthesis. In: Cane DE (ed) Comprehensive natural products chemistry: isoprenoids, including steroids and carotenoids. Elsevier, Oxford, pp 97–153CrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2016

Authors and Affiliations

  • Helena Trindade
    • 1
  • Inês Sena
    • 1
  • A. Cristina Figueiredo
    • 1
  1. 1.Centro de Estudos do Ambiente e do Mar Lisboa, Faculdade de Ciências da Universidade de LisboaDepartamento de Biologia Vegetal, Centro de Biotecnologia VegetalLisbonPortugal

Personalised recommendations