Nitrate reductase activity quantitatively predicts the rate of nitrate incorporation under steady state light limitation: A revised assay and characterization of the enzyme in three species of marine phytoplankton

Abstract

The enzyme nitrate reductase (NR) has been proposed as an index of nitrate incorporation rates in marine phytoplankton, but it has proven difficult to interpret NR measurements in field settings because many previous NR assays have been poorly optimized and NR activity in phytoplankton has been poorly characterized under steady state conditions. An NR assay was developed for the diatom Thalassiosira pseudonana using an extraction in phosphate buffer with Triton X-100, EDTA, dithiothreitol, polyvinyl pyrrolidone, and bovine serum albumin. NR activity in homogenates was stable for up to 1 h, but filtered samples could be stored for 96 h in liquid nitrogen without significant loss of activity. Addition of FAD to crude extracts of T. pseudonana had no effect, whereas the effect on desalted extracts or crude extracts from other species, varied from decreases in NR activity to over 250% increases. Half-saturation constants (K,) varied between species; high levels of NADH or nitrate were found to be inhibitory in some cases. These results indicate a wide diversity of forms of NR in marine phytoplankton.Under continuous, light-limited growth, NR activity was quantitatively related to calculated rates of nitrate incorporation (pN) in T. pseudonana, Skeletonema costatum, and three other diatom species examined. The relationship differed for 10 other species; NR activity was equal to bN in some cases, but higher or lower in others. In dinoflagellates, in particular, NR activity was highly correlated with pN, but accounted for ~20% of hN in Amphidinium carterae.The importance of nitrogen metabolism in general, and nitrate metabolism in particular, in the marine environment is based not only on the frequent identification of nitrogen as a nutrient limiting primary production, but also because of the important differences between the fate of production depending on whether newly available nitrogen (e.g. nitrate) or regenerated forms (e.g. ammonium) are used (Dugdale and Goering 1967;Platt et al. 1992). Thus, measurements of rates of nitrate incorporation (sensu Wheeler 1983, i.e. the combination of inorganic nitrogen into large macromolecules such as proteins and nucleic acids) by marine phytoplankton are critical to understanding rates of primary production and biogeochemical cycling in many areas of the oceans.Current methods of estimating rates of nitrate incorporation are based largely on incubation techniques with the stable isotope 15N as a tracer (Dugdale and Wilkerson 1986). It is appreciated that there are problems with this

Soluble and membrane‐associated nitrate reductases in the dinoflagellate Peridinium gatunense

“…This was clearly documented by biochemical and immunocytochemical data. In other studies, the enzyme was purified (Ramalho et al, 1995) or activity was determined in crude extracts (Berges & Harrison, 1995), but a soluble NR form was not mentioned. The present work definitely proves the existence of a soluble NR that carries out up to 97% of NO $ − reduction under optimal growth conditions.…”

Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress

“…They clearly are not driven by a developmental program but rather, responses to environmental cues. For example, under optimal growth conditions, ∼40% of the photosynthetically fixed carbon in typical eukaryotic microalga is directed toward the synthesis of amino acids that ultimately are incorporated into proteins (1)(2)(3). Over 50 y ago, however, it was recognized that, when nitrogen limits growth, intermediate metabolism is altered, and many microalgae can accumulate storage lipids, mainly in the form of triacylglycerols (TAGs) (4)(5)(6).…”

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

“…Nitrate is taken up into the diatom cell, where it is first reduced to nitrite by a cytosolic NADH-dependent nitrate reductase (NR; Gao et al, 1993;Berges and Harrison, 1995;Allen et al, 2005). Nitrite is then transported into the chloroplast and further reduced to ammonium by a cyanobacterium-like ferredoxindependent nitrite reductase (Fd-NiR; Milligan and Harrison, 2000;Bowler et al, 2010).…”