title: Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.
abstract: 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 plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. 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, and new hints to engineer eukaryote endomembranes for future biotechnological applications.
authors.affiliation: DiSTeBA (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy. Electronic address: gp.disansebastiano@unisalento.it.
authors.forename: Gian Pietro
authors.initials: GP
authors.lastname: Di Sansebastiano
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Rizzello
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
authors.forename: Miriana
authors.initials: M
authors.lastname: Durante
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
authors.forename: Sofia
authors.initials: S
authors.lastname: Caretto
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy; DII (Dipartimento di Ingegneria dell'Innovazione), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Nisi
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: De Paolis
authors.affiliation: DiSTeBA (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Faraco
authors.affiliation: DiSTeBA (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Montefusco
authors.affiliation: DiSTeBA (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali), University of Salento, Campus ECOTEKNE, 73100 Lecce, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Piro
authors.affiliation: CNR - ISPA (Istituto di Scienze delle Produzioni Alimentari), Via Monteroni, 73100 Lecce, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Mita
mesh_terms: D000962:Antimalarials / Q000378:metabolism; D031172:Artemisia annua / Q000254:growth & development* / Q000378:metabolism; D037621:Artemisinins / Q000378:metabolism*; D002451:Cell Compartmentation; D018929:Cell Culture Techniques; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007783:Lactones / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D005456:Fluorescent Dyes; D007783:Lactones; D010940:Plant Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C075875:artemisin
keywords: Artemisia annua; Cell compartmentalization; SNARE; SYP51; Vacuole