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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

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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

Analytical Method Information

ID M01
Method Details ID MS01
Sample Amount 10 µl

Analytical Method Details Information

Instrument Accela LCQ Fleet Ion trap (Thermo Finnigan)
Instrument Type UPLC-QTOF-MS
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.

Chromatography/mass spectrometry conditions
Funaria hygrometrica extracts were analyzed using an ultra performance liquid chromatography electrospray mass spectrometry (UPLC–ESI–MSn) system Accela LCQ Fleet Ion trap (Thermo Finnigan). The compound mixture was separated on a Hypersil Gold C18 column (50 × 2.1 mm, 1.9 µm particle size). 10 µl were injected per sample. The mobile phase consisted of H2O with 0.1% (v/v) formic acid (solvent A) and methanol with 0.1% (v/v) formic acid (solvent B). The solvent gradient program was as follows: 30% B, 0–0.5 min; 30–80% B, 0.5–6 min; 80–100% B, 6–24.9 min; 100–30% B, 24.9–25 min; 30% B, 25–30 min. The flow rate was 400 µl min−1. Column oven temperature was maintained at 35 °C. Spectra were acquired in full scan mode 50–1000 m/z range, operating in positive mode; the scan time was 500 ms (3 micro-scans). The ESI source parameters were set as follows: capillary temperature 320 °C; capillary voltage 20 V; spray voltage 4.5 kV; tube lens 40 V; nitrogen sheath gas 25 arbitrary units (AU); auxiliary gas 15 AU. For the MSn analysis, collision energy (CID) was normalized to 35.


Data Analysis Information

ID D01
Title Calculation of chemical sum formulas
Data Analysis Details ID DS01
Recommended decimal places of m/z

Data Analysis Details Information

Title Calculation of chemical sum formulas
Description For manually selected accurate mass spectra, the chemical composition of the respective signals was calculated. The program SpiderMass (Winkler 2015), which takes into account the m/z and isotopic distribution of MS signals, was used for formula generation and evaluation. The putative formulae were evaluated according to the criteria of the “Seven Golden Rules” (Kind and Fiehn 2007). Following, the results were ranked by their SpiderHit, which is based on the mass difference and isotope distribution deviation between measured and theoretical values. The following parameters were used: mass error 0.05 m/z, composition C/H/D/N/O, Isotope Distribution Fit (IDF) on.
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