Characterization of Artemisia annua compartmentalization to explore the possibility of endomembranes engineering.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion (Krause et al., 2013) thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of protoplasts derived from Artemisia suspension cultures and explore a novel strategy based on compartmentalization engineering using a modified SNARE protein as molecular tools. We used several fluorescent markers to visualize the vacuolar organization in vivo and a truncated form of AtSYP51, 51H3, to induce a compartment generated by the contribution of endocytosis and ER-to-Vacuole traffic. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE (De Benedictis et al. 2013), and new hits to engineer eukaryote endomembranes for future biotechnological applications. De Benedictis, M., Bleve, G., Faraco, M., Stigliano, E., Grieco, F., Piro, G., Dalessandro, G., Di Sansebastiano, G.P., 2013. AtSYP51/52 functions diverge in the Post-Golgi traffic and differently affect vacuolar sorting. Mol Plant. 6, 916–930. Krause, C., Richter, S., Knöll, C., Jürgens, G., 2013. Plant secretome - from cellular process to biological activity. Biochim Biophys Acta. 1834, 2429–2441.