SE167:/S01/M02

Sample Set Information

ID	TSE1329
Title	Funaria hygrometrica Hedw. elevated tolerance to D2O: its use for the production of highly deuterated metabolites.
Description	The method introduced here to grow F. hygrometrica in high concentrations of D 2 O is an excellent alternative to produce highly deuterated metabolites with broad applications in metabolic studies. Our mass spectrometry experiments strongly indicate the successful incorporation of deuterium into organic compounds.. This approach also has limitations as D2O in high concentrations negatively affects the survival of most organisms. Here we report the moss Funaria hygrometrica as an unusual high tolerant to D2O in liquid culture. We found that this moss is able to grow in up to 90% D2O, a condition lethal for many eukaryotes. Mass spectrometric analyses of F. hygrometrica extracts showed a strong deuteration pattern. The ability to tolerate high concentrations of D2O together with the development of a rich molecular toolbox makes F. hygrometrica an ideal system for the production of valuable deuterated metabolites.
Authors	Vergara, F., Itouga, M., Becerra, R.G., Hirai, M.Y., Ordaz-Ortiz, J.J., and Winkler, R.(2018)
Reference	Planta (2018) 247: 405. DOI: 10.1007/s00425-017-2794-5
Comment

https://link.springer.com/article/10.1007%2Fs00425-017-2794-5

Sample Information

ID	S01
Title	Funaria hygrometrica
Organism - Scientific Name	Funaria hygrometrica
Organism - ID	NCBI:txid29583
Compound - ID
Compound - Source
Preparation	Funaria hygrometrica was collected from reclaimed land in Omuta City, Fukuoka Prefecture, Kyushu, Japan (130°23′E, 33°1′N), in April 2003. The spores were sown on a modified Knop’s agar medium: 10 mM KNO3, 1 mM MgSO4, 2 mM KH2PO4, 10 mM CaCl2, 45 µM FeSO4, 1.6 µM MnSO4, 10 µM H3BO3, 0.2 µM ZnSO4, 0.2 µM KI, 0.1 µM Na2MoO4, 0.2 µM CuSO4, 0.2 µM CoCl2, 5 mM (NH4)2C4H4O6, pH 5.5, and 1% (w/v) agar in plastic Petri dishes (60 × 15 mm). Cellophane (PL-#300, Futamura Chemical Industries Co., Ltd., Japan) washed with 5 mM EDTA·4Na and MilliQ water was placed on the media before sowing. Protonema cells were grown under fluorescent light at 80 µmol m-2 s-1 with a 16/8 h light/dark cycle at 23 °C. F. hygrometrica protonema cells grown on the agar were collected and suspended in the modified Knop’s liquid medium using a polytron (PT-MR2100, Kinematica, Switzerland) operated at minimum speed for 15 s. The suspended cells were inoculated with 0.5 l liquid medium in a culture bottle (Fujimoto Scientific Co., Ltd., Cat. no. XX151), and cultured with aeration at 1000 ml min-1 under fluorescent light at 80 µmol m-2 s-1 with a 16/8 h light/dark cycle at 23 °C. The growth rate of the culture was examined by measurement of the dry weight of cells for 2 weeks. For the isotopic labeling protonemal (juvenile stage of the gametophyte phase) cells of F. hygrometrica were cultured in modified Knop’s liquid medium (see above). This culture medium was separately prepared in six different concentrations of D2O in H2O: 0, 10, 20, 40, 50, 70 and 90% of D2O. The pH was adjusted in each case to 5.5 and 0.5 l of each medium was poured into Roux culture bottles. Air was injected into the media by bubbling at a rate of 1 l min-1 and light was provided by fluorescent lamps at 80 µmol m-2 s-1 with a 16/8 h light/dark cycle. Temperature was maintained at 23–25 °C. F. hygrometrica protonemal cells were first inoculated in the medium with 0% D2O and after the culture reached cell saturation aliquot was transferred to the medium with 10% D2O. This process was systematically repeated to gradually transfer F. hygrometrica protonemal cells to the immediately following higher concentration of D2O and up to reaching 90% D2O.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M02
Title	Non-targeted metabolite profiling
Method Details ID	MS02
Sample Amount	10 µl
Comment

Analytical Method Details Information

ID	MS02
Title	Non-targeted metabolite profiling
Instrument	LC: Acquity UPLC System (Waters, Milford, USA) , MS: QTOF Synapt G1 (Waters, UK)
Instrument Type
Ionization	ESI
Ion Mode	positive
Description	Plant metabolites extraction After cell saturation was achieved F. hygrometrica cultures were transferred to plastic containers, flash-frozen with liquid nitrogen and subsequently freeze-dried. Dried plant material was ground with a Mixer Mill MM 301 (Retsch®) at 30 Hz for 15 s. Plant powder (20 mg of every sample) was extracted with 1.5 ml of HPLC grade methanol for 25 min in ultrasound bath at room temperature. Extracts were centrifuged at 15,870g for five min at room temperature and the supernatants were filtered using a 0.2 µm nylon syringe filter. Filtrates were subjected to UPLC–MSn analysis. Non-targeted metabolite profiling The samples extracted with methanol were reconstituted in a mixture of methanol/deionized water/formic acid [75:24.85:0.15 (v/v/v)] and filtered through a 0.2-μm filter. Chromatographic separation was achieved on an Acquity UPLC System (Waters, Milford, USA) using a CSH C18 2.1 × 150 mm, 1.7 µm column maintained at 40 °C. The samples were injected (10 μl) and elution of compounds was performed at a flow rate of 0.3 ml min−1, the gradient comprised mobile phase A: deionized water containing 0.1% formic acid; and mobile phase B: acetonitrile containing 0.15% formic acid. The gradient program was isocratic for the first 30 s, then a linear gradient increase from 30 to 80% of solvent B at 6 min, 100% of B at 24.9 min, and hold for 1 min for column washing and 4 min at 30% of solvent B for column re-equilibration. The mass spectrometer comprised an orthogonal QTOF Synapt G1 (Waters, UK) operated under the following conditions: positive electrospray ionization mode, capillary voltage at 3.0 kV, cone voltage 45 V, extractor voltage 4.0 V, source and desolvation temperature were 150 and 350 °C, respectively. Cone and desolvation gas flow was nitrogen at a flow rate of 20 l h−1 and 700 l min−1. Leucine-enkephalin (M + H)⊕ = 556.2771 was infused at a flow rate of 5 μl min−1 at concentration of 2 ng ml−1 during acquisition as internal mass calibrant. MS data were acquired on continuum mode and processed with MassLynx (version 4.1, Waters, USA) and Progenesis QI for small molecules (Nonlinear Dynamics version 2.3, Waters, USA) software, using Progenesis MetaScope Database, with Biomolecules, LipidsMAPS, and LipidBlast as searching parameters, accepting precursor tolerance of 10 ppm for putative identifications.
Comment_of_details