Development of Approaches for Deuterium Incorporation in Plants

Soon after the discovery of deuterium, efforts to utilize this stable isotope of hydrogen for labeling of plants began and have proven successful for natural abundance to 20% enrichment. However, isotopic labeling with deuterium (²H) in higher plants at the level of 40% and higher is complicated by both physiological responses, particularly water exchange through transpiration, and inhibitory effects of D₂O on germination, rooting, and growth. The highest incorporation of 40-50% had been reported for photoheterotrophic cultivation of the duckweed Lemna. Higher substitution is desirable for certain applications using neutron scattering and nuclear magnetic resonance (NMR) techniques. ¹H²H NMR and mass spectroscopy are standard methods frequently used for determination of location and amount of deuterium substitution. The changes in infrared (IR) absorption observed for H to D substitution in hydroxyl and alkyl groups provide rapid initial evaluation of incorporation. Short-term experiments with cold-tolerant annual grasses can be carried out in enclosed growth containers to evaluate incorporation. Growth in individual chambers under continuous air perfusion with dried sterile-filtered air enables long-term cultivation of multiple plants at different D₂O concentrations. Vegetative propagation from cuttings extends capabilities to species with low germination rates. Cultivation in 50% D₂O of annual ryegrass and switchgrass following establishment of roots by growth in H₂O produces samples with normal morphology and 30-40% deuterium incorporation in the biomass. Winter grain rye (Secale cereale) was found to efficiently incorporate deuterium by photosynthetic fixation from 50% D₂O but did not incorporate deuterated phenylalanine-d8 from the growth medium.