Nitrate reductase and urease enzyme activity in the marine diatom Thalassiosira weissflogii (Bacillariophyceae): interactions among nitrogen substrates

The dissolved nitrogen pool in aquatic systems is comprised of many different nitrogen forms, both inorganic and organic. Interaction among these nitrogen forms at the level of uptake and enzyme activity is, with the exception of NH4 + and NO3 , not completely understood. Nitrate reductase (NR) and urease (UA) activities in the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle were measured in NO3 , NH4 , and urea-sufficient cultures before and after challenge additions of NH4 , NO3 , and urea in a factorial design. NR and UA were constitutively expressed during growth on NO3 , NH4 , and urea. Growth on NH4 + or urea resulted in NR activities that were <10% of the activity observed in the NO3 -grown culture, while growth on NO3 ) resulted in UA values that were 35% of the activities during growth on either NH4 + or urea. The addition of NH4 + or urea to NO3 -grown cultures resulted in an immediate decrease in cellular NO3 ) uptake rate, which was not mirrored by an immediate repression of in vitro NR activity; however, the diel peak in NR was suppressed in these challenge experiments. The addition of NO3 ) or NH4 + to urea-grown cultures resulted in non-significant decreases in the urea uptake rate. UA was not impacted by NO3 ) addition, but NH4 + addition significantly decreased UA throughout the experiment. These studies demonstrate that the uptake and assimilation of NO3 ) and urea may not be subject to the same internal feedback mechanism when challenged with other nitrogen substrates. Introduction Dissolved nitrogen in aquatic systems exists in several different forms, NO3 , NO2 , NH4 , and dissolved organic nitrogen (DON), simultaneously, all of which have been shown to support active phytoplankton growth (see reviews by Antia et al. 1975, 1991). There are two distinct steps in the conversion of these extracellular dissolved nutrients into particulate biomass (i.e. new cells), nutrient transport into the cells and the assimilation of these nutrients into proteinaceous material (e.g. McCarthy 1981; Syrett 1981; Flynn 1998). It is often difficult to study nutrient transport independent of assimilation, so nutrient transport experiments commonly use non-metabolizable substrates, e.g. radiolabelled chlorate is used to study the uptake of NO3 ) (Falkowski 1975; Balch 1987). These surrogate substrates, however, are not assimilated and may lead to differences in the regulation of nutrient uptake when compared to the metabolically active substrate. Nitrogen isotope incubations may also represent a combination of transport and assimilation depending upon post-transport feedback processes (e.g. Glibert and Goldman 1981; Goldman and Glibert 1982; Collos 1983; Flynn 1998). Importantly, these isotopic incubations measure nutrient transport rates under incubation conditions and reflect in situ rates only if conditions resemble those in the field. This pitfall may be avoided by measuring enzyme activities (Berges and Harrison 1993), although enzyme activity measurements themselves are subject to potential problems such as nonoptimized assay conditions or instability of the enzyme following extraction from the cell. Moreover, internal substrate concentrations at the time of sample collection are usually not measured, and therefore assays yield at best potential rates. Despite this, enzymes should serve as integrators of past nutrient assimilation history over time scales comparable to growth rates under nitrogensufficient conditions (e.g. Berges and Harrison 1993, 1995). Within the past decade, significant improvements in optimizing enzyme assays in marine diatoms have Marine Biology (2004) 144: 37–44 DOI 10.1007/s00227-003-1181-x