The identification, isolation and functional characterization of two genes encoding two monoterpene synthases-γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)-from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon-intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time-course study of the essential oils (EOs) of 2-year-old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.

The essential oils of Thymus caespititius, T. camphoratus, T. capitellatus, T. carnosus, T. pulegioides, T. zygis subsp. zygis and T. zygis subsp. sylvestris collected in diff erent regions of Portugal, including the Azores islands, were evaluated for preventing lipid peroxidation and scavenging free radicals, and hydroxyl and superoxide anions. T. zygis subsp. zygis oil possessed the best capacity for preventing lipid peroxidation (IC 50 = 0.030 ± 0.003 mg/ml), immediately followed by the oils isolated from T. zygis subsp. sylvestris collected in diff erent regions of Portugal. IC 50 values ranged from 0.066 ± 0.003 to 0.087 ± 0.001 mg/ml in oils isolated from samples from Alcanena and Duas Igrejas, respectively. The oils isolated from T. zygis subsp. sylvestris, independent of the harvesting region, were shown to be the most eff ective for scavenging free radicals (0.358-0.453 mg/ml). The best capacity for scavenging hydroxyl radicals was found in the oils of T. camphoratus collected in Espartal and of T. caespititius of Lordelo, with IC 50 < 0.5 mg/ml. The low oil amount did not allow IC 50 values for the superoxide anion scavenging determination, therefore considering a unique oil concentration (5 mg/ml), the highest inhibition percentages (>50%) were registered with the essential oils of T. zygis subsp. sylvestris from Duas Igrejas and Covão do Coelho, and of T. capitellatus from Sines.

The antioxidant and antiproliferative activities of the essential oils from Thymbra capitata and Thymus species grown in Portugal were evaluated. Thymbra and Thymus essential oils were grouped into two clusters: Cluster I in which carvacrol, thymol, p-cymene, α-terpineol, and γ-terpinene dominated and Cluster II in which thymol and carvacrol were absent and the main constituent was linalool. The ability for scavenging ABTS•+ and peroxyl free radicals as well as for preventing the growth of THP-1 leukemia cells was better in essential oils with the highest contents of thymol and carvacrol. These results show the importance of these two terpene-phenolic compounds as antioxidants and cytotoxic agents against THP-1 cells.