Team:Valencia UPV/Project/results/pheromone analysis

It has been a hard way to obtain the first results. Summarizing: the different parts needed to build the pheromone biosynthesis device were domesticated and assembled with the help of GoldenBraid 2.0 following the flowchart shown in the Mehtodology section. Once the constructs were obtained, they were transiently transformed by agroinfiltration into our plant chassis, N. benthamiana. At this point, it was time to check if the plant was actually producing the target pheromones.

With this objective, the agroinfiltated plants were analysed for pheromone production using HS-SPME coupled to GC-MS. After the analysis we obtained different chromatograms to prove it. This is the volatile profile of a normal Nicotiana benthamiana leaf, the control of the experiment (Figure 1):

On the other hand, Figure 2 shows the volatile profile of the modified one. As it can be appreciated at first sight, there are two additional peaks in the transformed plants which are not present in the control. The mass spectrum of the molecules corresponding to each peak was compared with the NIST database, which confirmed that both molecules were the desired pheromones, (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate (see biosynthesis). In addition, to have an unquestionable validation we performed a GC-MS analysis of the standards, chemically synthetized pheromones provided by the C.E.Q.A. They corresponded with the obtained peaks, both the retention time and the mass spectrum.

As it can be observed, not only the production of the pheromones was achieved, but also they were among the most abundant volatile compounds in the plant. In addition, as it can be appreciated, the ratio between the abundance of (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate was approximately 4 to 1. This means that unprecedented conversion rates were achieved, compared to previous publications (Ding et al. 2014). Such improved conversion rate can be attributed??? to the use of a single multigene construction (see Results- Constructs- Biosynthesis) comprising all three genes needed to produce the pheromones in a single plasmid and thus, facilitating the simultaneous expression of the three enzymes in the plant cells. (see Biosynthesis).

Finally, even though the obtention of the enzyme necessary for the biosynthesis of (Z)-11-hexadecenal was not possible (see notebook: FAO1 obtention), the possible presence of this pheromone was also checked. Therefore, the standard was analysed and the corresponding molecule was looked for in the transformed N. benthamiana leaf chromatogram. Surprisingly, a small peak was identified, that was not present in the control and corresponded to (Z)-11-hexadecenal. The conversion of (Z)-11-hexadecen-1-ol into (Z)-11–hexadecenal was probably performed by an endogenous alcohol oxidase from the plant, which was documented as not having significant in vivo activity (Sol Genomics Network). As the conversion rate was low, this opened a field for futher studies on pheromone plant-production.

At the end of the journey we have achieved a plant able to produce three insect sexual pheromones:

• (Z)-11-hexadecen-1-ol as the most abundant plant volatile.
• (Z)-11-hexadecenyl acetate as one of the most abundant volatile molecules in the plant, with an unprecedented conversion yield.
• (Z)-11-hexadecenal, produced by an endogenous plant enzyme at? low yield that could be improved in future studies.