The dissertation research investigated the cellular and molecular mechanisms that regulate nitrate assimilation, the major pathway by which marine phytoplankton acquires nitrogen (N) from the environment. Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, a rate-limiting step in nitrate assimilation. As a key regulatory component in nitrate assimilation, NR from a marine diatom, SKELETONEMA COSTATUM, was targeted for investigation. The biochemical and regulatory features of the diatom NR were investigated and the applicability of NR abundance as an index for estimating new production was explored.

Biochemical characterization using both purified NR and cell-free extracts revealed several unique features of the enzyme quite distinct from vascular plant NRs. The features included the molecular mass of native enzyme, heavy metal sensitivities of the activity and temperature dependence of the enzyme activity and abundance. The polyclonal antibodies generated against purified diatom NR were demonstrated to be specific for diatoms. The antigenicity of NRs from marine phytoplankton are divergent. These features alone provided significant insights and entrees into the central role of NR in controlling the production dynamics of phytoplankton in the ocean.

Environmental impacts on the initial events in nitrate assimilation (nitrate transport and reduction) were investigated by monitoring NR activities, NR protein abundance and internal nitrate levels determined by a rapid method developed in this research. The results demonstrated the independent environmental controls on nitrate transport and reduction. Long-term exposure to ammonium completely eliminates nitrate assimilation capacity. However, induction of nitrate transport and reduction capacities by nitrate and light is rapid, requires both light and nitrate, and involves de novo synthesis of NR protein and a nitrate transport system. Further, it is concluded that internal nitrate and redox state associated with photosynthetic electron transport may provide a regulatory signal essential for the induction. It was also demonstrated that ammonium inhibition of nitrate assimilation is at the level of nitrate transport rather than nitrate reduction and that ammonium and nitrate assimilation can occur concomitantly providing that internal nitrate pools are adequate.

The use of antibodies to detect NR protein in intact phytoplankton cells and to trace NR abundance in response to environmental shifts were explored. It was demonstrated that NR protein is not constitutively expressed, its abundance is sensitive to environmental shifts, and it may be a better index than in vitro activity for estimating nitrate assimilation potential. Further, a protocol for in situ detection of NR was developed, which allows for detecting NR protein in individual cells by flow cytometry and resolving fine scale dynamics of NR protein abundance in response to environmental transitions.

This research has not only significantly advanced our understanding of the underlying biological mechanisms that control the production dynamics of marine phytoplankton, but has also opened a variety of research opportunities in NR biochemistry, phylogeny, eco-physiology and molecular biology. In addition, the present investigation demonstrated the significance of biochemical and molecular approaches and subcellular features of marine organisams for obtaining mechanistic understandings of biological processes in the ocean.