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Funaria hygrometrica was collected from re … 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. tration of D2O and up to reaching 90% D2O.
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