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

Abstract

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).

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Abbreviations

3′-UTR:

3′-Untranslated region

5′-UTR:

5′-Untranslated region

aa:

Amino acid

CTAB:

Cetyl trimethyl ammonium bromide

DEPC:

Diethyl pyrocarbonate

dNTPs:

Deoxynucleotides

DTT:

1,4-Dithiothreitol

FDP (also known as FPP):

Farnesyl diphosphate (also known as farnesyl pyrophosphate)

GC:

Gas chromatography

GC–MS:

Gas chromatography–mass spectrometry

GDP (also known as GPP):

Geranyl diphosphate (also known as geranyl pyrophosphate)

LB:

Luria–Bertani medium

ORF:

Open reading frame

PCR:

Polymerase chain reaction

PMSF:

Phenylmethylsulfonyl fluoride

PpAPS:

Pinus pinaster α-pinene synthase

PpnAPS:

Pinus pinea α-pinene synthase

PWD:

Pine wilt disease

PWN:

Pinewood nematode (Bursaphelenchus xylophilus)

RT:

Reverse transcription

SH:

Schenk and Hildebrandt culture medium

SPME:

Solid phase micro extraction

WV5:

Westvaco culture medium

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Acknowledgments

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.

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Correspondence to A. Cristina Figueiredo.

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H. Trindade and I. Sena equally contributed to this work.

Communicated by E. Kuzniak-Gebarowska.

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Trindade, H., Sena, I. & Figueiredo, A.C. Characterization of α-pinene synthase gene in Pinus pinaster and P. pinea in vitro cultures and differential gene expression following Bursaphelenchus xylophilus inoculation. Acta Physiol Plant 38, 143 (2016). https://doi.org/10.1007/s11738-016-2159-x

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Keywords

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