Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO3 to NO2. A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S.costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS NR protein could be detected in NO3‐grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH4‐grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological “steady‐state” 96 hrs later. NR activity exhibited diel variation with maxima at mid‐day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post‐translational activation of NR protein. CONCLUSIONS The presented protocol allows the differentiation of NH4‐ versus NO3‐grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities. Cytometry 39:173–178, 2000 © 2000 Wiley‐Liss, Inc.