Nicotiana benthamiana, Domin

Andersen, Trine Bundgaard, Rasmussen, Silas Anselm, Christensen, Søren Brøgger & Simonsen, Henrik Toft, 2019, Biosynthesis of tovarol and other sesquiterpenoids in Thapsia laciniata Rouy, Phytochemistry 157, pp. 168-174 : 169

2.3. Characterization of TlTPS509 expressed in N. benthamiana

HS-SPME-GC-MS analysis of N. benthamiana leaves expressing Tl TPS509 resulted in the detection of two major sesquiterpene alcohols, guaiol (3) (RI: 1585, 34% of the area) and bulnesol (4) (RI: 1654, 20% of the area) ( Fig. 3 ). Compound 3 and 4 exhibited similar EI MS spectrum to those of bulnesol and guaiol in NIST and Wiley GC-MS spectrum libraries. The product profile showed several additional compounds, and we could identify two minor products; guaiene (RI 1418, Rt: 8.6)) and bulnesene (RI 1487, Rt: 9.18) ( Fig. 3 ). Several peaks with a m/z of 220 were also observed but not identified. A hexane extract of N. benthamiana leaves, expressing Tl TPS509 was analyzed by GC-MS using a Programmed Temperature Vaporising injector (PTV) port. By using a low initial temperature of 60 ̊C followed by a gradual increase to 250 ̊C it was expected that thermal rearrangements were avoided. Again, 3 and 4 were detected as major compounds, but not guaiene and bulnesene, which were probably below the detection limit. Furthermore, neither of the unknown compounds with m/z 220 were detected in the PTV analysis.

Figures

Fig. 3. HS-SPME GC-MS analysis of TlTPS509 expressed together AtHMGR, the control shows the expression of AtHMGR alone. Guaiol (3) is seen as the major product (Rt 9.89 min), with bulnesol (4), Rt: 10.2, a-guaiene (Rt: 8.6), and a-bulnesene (Rt: 9.18) as minor products. * show unidentified sesquiterpene like products, which is either ketone formed during the biosynthesis or breakdown products of the alcohols formed during the GC-MS analysis. Based on this TlTPS509 is a guaiol synthase. MS spectra are presented in supplementary data.