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Physiological and environmental controls on the nitrogen and oxygen isotope fractionation of nitrate during its assimilation by marine phytoplankton

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Karsh, KL (2014) Physiological and environmental controls on the nitrogen and oxygen isotope fractionation of nitrate during its assimilation by marine phytoplankton. PhD thesis, University of Tasmania.

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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 outside the cell by efflux. They also provided the first step in
establishing how the 18εorg:15εorg near 1 for nitrate assimilation originates at the cellular level.
Finally, the whole cell N and O isotope effects (15εorg and 18εorg) for nitrate
assimilation were measured in steady state cultures of T. weissflogii to assess how
environmental and physiological parameters affected the extent of nitrate efflux and thus the
magnitude of 15εorg and 18εorg. Steady state cultures ensured 15εorg was not affected by
transients in culture conditions and that 15εorg could be related to intracellular nitrate
concentration and N and O isotopic composition. As observed in previous studies, 15εorg and
intracellular [NO3
-] increased under light limitation (a 3-fold and 5-fold increase respectively, relative to results in non-limited cultures). 15εorg and intracellular [NO3
-] were invariant under
phosphate limitation. The ratio 18εorg:15εorg was near 1 under all conditions. In conjunction
with previous results from iron- and temperature-limited batch cultures, these results suggest
the N and O isotope effects for nitrate assimilation are (i) invariant under most environmental
conditions and that (ii) irradiance may be the major driver of variability in 15εorg and 18εorg in
the ocean.
The measurements also yielded insight into the regulation of nitrate efflux and
suggest a role for intracellular NO3
- storage in the environment. The near-constant magnitude
of 15εorg observed under phosphate-limited growth suggests that the ratio of nitrate efflux to
uptake remains 12-15% across a 4-fold increase in growth rate. The constant, non-zero efflux
rate this implies suggests a component of efflux is inevitable and correlated with growth rate.
The high 15εorg measured under light limitation show that efflux rates increase to 60% of
gross nitrate uptake rates, suggesting that an additional component of efflux is correlated with
high intracellular [NO3
-]. The high intracellular [NO3
-] observed under light limitation may
suggest an adaptation to rapidly changing irradiance in the turbulent mixed layers of the
surface ocean: excess intracellular NO3
- storage may maximize assimilation rates and/or
provide an electron sink to dissipate excess energy.

Item Type: Thesis (PhD)
Keywords: Nitrogen, nitrate, isotope, marine, algae, assimilation
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Date Deposited: 17 Nov 2014 01:28
Last Modified: 15 Sep 2017 01:06
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