Thujopsene

Arabidopsis seedlings were grown on AM and poplar plantlets on P20 medium in one half of the bi-compartmented Petri dishes, as described above. The adjacent compartment was equipped with sterile filter paper (1 × 1 cm; Whatman, Maidstone, UK). (–)-Thujopsene (≥97%; Sigma) and β-caryophyllene (≥80%; Sigma) were diluted to final concentrations of 1, 10, 100 and 1,000 p.p.b. in n-pentane (HPLC grade, Carl Roth GmbH & Co. KG). Thirty microlitres of the solution was dropped on the filter paper. The control plates were equipped with 30 μl of n-pentane. The treatment was performed once at the beginning of the experiment. The Petri dishes were immediately sealed with Parafilm and the plants were grown under controlled environmental conditions as described before. The LRs were quantified after 0, 4, 7 and 10 days of treatment. VOCs were collected for 6 h after 3, 7 and 10 days. Because no significant changes of the VOCs patterns were observed, only data for 10 d.h.c.c. are shown. Furthermore, Arabidopsis seedlings or poplar plantlets were grown in bi-compartmented plates with or without L. bicolor with lovastatin, as described above. The compartment with the inhibited fungus was supplemented with 30 μl of 100 p.p.b. (–)-Thujopsene solution or n-pentane as the control. The LRs were quantified after 10 days (n=5–10 plates per treatment).

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and phosphate-buffered saline (PBS) for cell culture were purchased from Gibco-BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS), thiazolyl blue tetrazolium bromide (MTT), Griess reagent, sodium nitrite solution, the 19 selected volatile compound standards (α-pinene, camphene, β-pinene, sabinene, 3-carene, β-myrcene, α-phellandrene, α-terpinene, limonene, p-cymene, terpinolene, 2-nonanone, acetic acid, citronellal, bornyl acetate, thujopsene, β-chamigrene, (-)-β-bisabolene, and nerolidol) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). In addition, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). Antibodies against iNOS, p65, phospho-p65 (p-p65), and β-actin were obtained from Santa-Cruz Biotechnology (Dallas, TX, USA). Materials for western blot were obtained from Bio-Rad (Hercules, CA, USA), and all other reagents and chemicals used were of analytical grade.