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

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).