AbstractsBiology & Animal Science

Abstract

Nitrogen (N) is an essential nutrient for phytoplankton growth and its availability often limits primary production in the surface ocean. Quantifying the inputs, losses, and internal cycling of N is thus a major goal in marine biogeochemical research. The N isotope effect (15εorg) for nitrate assimilation is a key parameter in using N isotope distributions (15N/14N) to study the marine N cycle. Unexplained variability in its magnitude is a major source of uncertainty to N isotopic studies in the modern and past ocean. The ratio of the oxygen (O) and N isotope effects for nitrate assimilation (18εorg:15εorg) is also an important parameter; the association of nitrate assimilation with an 18εorg:15εorg near 1 is the cornerstone of studies using coupled nitrate N and O isotope measurements to separate co-occurring N cycle processes that have counteracting effects on the N isotopes alone. The association of nitrate assimilation with an 18εorg:15εorg near 1 is based on empirical evidence without full understanding of the physiological mechanisms generating the ratio. A better understanding of the controls on both the magnitude and ratio of N and O isotope effects for nitrate assimilation would strengthen environmental application of N and O isotopes. Towards this goal, the individual N and O isotope effects for each fractionating step in nitrate assimilation (nitrate reduction, uptake, and efflux) were measured. The reduction of nitrate to nitrite is catalyzed by the intracellular enzyme eukaryotic assimilatory nitrate reductase (eukNR). An N isotope effect of 26.6 ± 0.2‰ and nearly equivalent N and O fractionation were measured in two distinct forms of eukNR (the NADPH form in cell-free extracts from the fungus Aspergillus niger and the NADH form in cell homogenates from the marine diatom Thalassiosira weissflogii), suggesting these values will apply to the eukNR family as a whole. These are the first reliable N and O isotope effect measurements for an enzyme that catalyzes the rate-limiting step in nitrate assimilation for all eukaryotic plants and algae. The N and O isotope effects for nitrate uptake and efflux were measured in the marine diatom T. weissflogii. Nitrate uptake and efflux were isolated from nitrate reduction by growing the cells in the presence of tungsten, which substitutes for molybdenum in assimilatory nitrate reductase, yielding an inactive enzyme. The N isotope effects for nitrate uptake and efflux were 2.0 ± 0.3‰ and 1.2 ± 0.4‰, respectively. The O isotope effect was 2.8 ± 0.6‰ for both uptake and efflux, yielding ratios of O to N isotopic fractionation greater than 1 for both processes. In sum, these results confirmed the existing physiological model for isotopic fractionation during nitrate assimilation where the isotope effect associated with intracellular nitrate reductase is high, the isotope effect associated with nitrate uptake is low, and the magnitude of 15εorg depends on the degree to which intracellular fractionation by nitrate reductase is expressed…