Characteristics of Nitrogen- and Iron-Limited Growth in the Diatom Phaeodactylum tricornutum, and in Natural Phytoplankton Populations.
The biochemical pathways involved in nitrogen (N) utilisation by marine phytoplankton have received considerable attention over the last forty years, but our understanding of these processes, and how they are affected by environmental change is still far from complete. This study investigates N metabolism in marine phytoplankton in both a controlled laboratory environment (using the coastal marine diatom Phaeodactylum tricomutum), and in the open ocean (e.g. Jellicoe Channel and the Subtropical Convergence Zone, New Zealand). Although the characteristics of ammonium uptake have been extensively studied in marine phytoplankton, comparatively little information exists on rates of assimilation. In this study, a robust method for measuring the rate of ammonium assimilation after a transient addition of ammonium is described. The method relies on the measured ability of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to release unassimilated ammonium from the cell and prevent further assimilation. There was little or no correspondence between the rate of ammonium assimilation and rates of ammonium uptake or maximum glutamine synthetase (GS) activity in Phaeodactylum tricomutum. Moreover, in N-limited cells maximum GS activity was a poor measure of N incorporation under steady-state conditions. However, GS activity did provide reliable information on N status (e.g. increased GS activity with increased N-limitation). Comparisons of the effects of varying N-source suggest that nitrate-grown cells are not disadvantaged under conditions of N-limitation due to the extra costs associated with nitrate reductase (NR) and nitrite reductase (NiR) activity. The metabolic costs of growth on nitrate may be significantly increased under iron (Fe)-limitation, as both NR and NiR require Fe. Fe-limited chemostat cultures excreted nitrite and ammonium when grown on nitrate. This release is probably a response to insufficient photoreductant under Fe-limited conditions. However, under Fe-limitation cellular N and C was similar to that of Fe-replete cells, suggesting that the N-source used for growth (nitrate or ammonium) did not influence N-assimilation (i.e. that nitrate-grown cells were able to secure the extra reductant required to support growth) under Fe-limited, light saturating conditions. The Gln:Glu ratio (an index of the cellular N-status) was significantly reduced under N-limitation, but not under Fe-limitation. Measurement of several biochemical indicators of the physiological state of phytoplankton cells (e.g. Gln:Glu ratio, GS activity, and Fv/Fm ratio) permitted the nutrient status of phytoplankton populations to be investigated during the NIWA Ocean Fronts programme over the Subtropical Convergence Zone, New Zealand. Low Gln:Glu ratios suggested that phytoplankton in both Subtropical and Subantarctic waters were N-limited, with a marked increase in this ratio when Fe was added to Subantarctic phytoplankton. The temporal utilisation of N by neritic phytoplankton was also investigated in Jellicoe Channel, northeastern New Zealand. Again, several biochemical indicators (e.g. Gln:Glu ratio, GS activity, and Fv/Fm ratio) were used to identify the N-status of this neritic phytoplankton assemblage both during bloom and non-bloom periods.
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