Nitrogen assimilation in the marine diatom Phaeodactylum tricornutum

The phytoplankton form the largest group of marine plants. Animal productivity in the oceans is dependent on phytoplankton biomass. Nitrogen is a limiting nutrient and is usually taken up in the form of nitrate. However, there has been very little investigation of the assimilation of nitrate into the aminoacids of phytoplankton. The pathway of nitrate assimilation in the marine diatom Phaeodactylum tricornutum was investigated. Nitrate is reduced to ammonia by the enzymes nitrate reductase and nitrite reductase. Nitrate reductase was purified to homogeneity (as judged by gel-electrophoresis) by affinity chromatography. The enzyme had an apparent molecular weight of 301,000 and was made up of six sub-units. NADH and reduced flavin acted as donors to the purified enzyme. However, the low Km app for NADH suggests that NADH is the physiological donor. Ammonia formed during nitrate assimilation can be incorporated into glutamate via the glutamate synthase cycle or via glutamate dehydrogenase. Though various assay methods were used, glutamate dehydrogenase was not detected in P. tricornutum even when grown in ammonium chloride. The enzymes which make up the glutamate synthase cycle i.e. glutamine synthetase and glutamate synthase were detected in P. tricornutum grown in nitrate. These enzymes were purified using gel-filtration and ion-exchange chromatography. The apparent molecular weights of glutamine synthetase and glutamate synthase were 280,000 - 360,000 and 282,000 respectively. No other detailed investigation of these enzymes has been reported from any marine plant. Glutamine synthetase appeared to consist of two active forms (GS I and GS II) and, unlike in higher plants, it appeared that the proportion of GS I to GS II depended on the pH and the levels of magnesium and dithiothreitol present in the buffer. Glutamine synthetase had a high affinity for hydroxylamdne, a non-physiological substitute for ammonia. When P. tricornutum was grown in ammonium chloride, the activity of glutamine synthetase decreased and only GS I was found. Glutamate synthase was able to use only NADH as the electron donor. Several assay methods were tried but no methyl-viologen or NADPH-dependent activity was detected in either the crude extract or the purified enzyme. Ferredoxin from spinach which was reduced using dithionite, did not act as a donor to glutamate synthase. Glutamate synthase had a high affinity for NADH and glutamine. It was concluded that the glutamate synthase cycle was the main pathway for ammonium assimilation in the marine diatom P. tricornutum.