Nitrate reductase is known to be the enzyme regulating the reduction of nitrate to nitrite in plants. This reduction is the first and rate-limiting step in the transformation of inorganic nitrate to a cellular nitrogen form. Since this reduction process is essential to phytoplankton growth when nitrate is the only available nitrogen source, the species with the highest capability to reduce nitrate, or the highest nitrate reductase activity, should be the species most favored to dominate a phytoplankton population under nitrate-limiting conditions. To study this hypothesis, the nitrate reductase activities of two species were studied; these species are co-occurring yet dominate under different conditions. Thalassiosira nordenskioldii is the dominant species in Auke Bay, Alaska in early spring when nitrate levels are decreasing from 15 μM to 1 μM, and the temperature is about 5 C. Skeletonema costatum is dominant in mid-summer when nitrate levels are below 1 μM and the temperature is above 12 C. The results show that at 15 C, Skeletonema has a higher enzyme activity at all nitrate levels than does Thalassiosira, which is consistent with the hypothesis. In addition, Thalassiosira shows a higher enzyme activity at 10 C, nearer its optimal temperature for growth, than at 15 C, suggesting that temperature affects species succession through its influence on enzyme activity. The applicability of Michaelis-Menten kinetics to this reduction reaction, based on the enzyme activities measured for these two species, is doubtful but inconclusive. The results also have implications for such concepts as nitrate-limited growth and the Redfield model relating nutrient, O₂ and CO₂ changes in the ocean. / Graduation date: 1971

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The commencement of in vitro differentiation is mediated by genetic changes that result in selective expression of genes and a shift in metabolism. The role of nitrate reductase, a key enzyme of nitrate assimilation, during differentiation was examined in this study using an in vitro Nicotiana tabacum (tobacco) callus culture system. In particular, the effects of nitrogen and light/dark regimes on callus differentiation and nitrate reductase were investigated. Methodology required for the analysis of nitrate reductase regulation during in vitro tobacco callus differentiation was established. Optimised in vivo, in situ and in vitro nitrate reductase assays yielded similar values and patterns during tobacco callus culture development, and the in vivo assay was selected for nitrate reductase activity measurement during subsequent experiments. Western blot analysis of tobacco callus acetone-extracted protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a spinach polyclonal nitrate reductase antibody yielded major bands at 71 and 48 kD, with numerous minor bands. Extraction of callus protein in the presence of various protectants did not prevent cleavage of putative nitrate reductase polypeptide. Slot blot detection of nitrate reductase mRNA using a [32p]- labelled nitrate reductase cDNA probe isolated from the plasmid pBMC102010 was not possible due to non-specific binding to nitrocellulose filters. Northern blotting of RNA fractionated by agarose gel electrophoresis using a [32p]-labelled nitrate reductase cDNA probe identified a single mRNA species at 3.5 kb, the expected size of tobacco nitrate reductase mRNA. In vitro tobacco callus differentiation on 60 or 120 mM nitrogen regimes and under light/dark (16/8 h), continuous dark or continuous light treatments were comparable in terms of fresh weight, protein and nitrate uptake. Higher levels of in vivo nitrate reductase activity were observed prior to visible shoot primordia in all treatments, suggesting that the developmental status of callus mediated the regulation of nitrate reductase. Putative nitrate reductase protein levels were not correlated with in vivo nitrate reductase activity during initial stages of tobacco callus differentiation under various light treatments; nitrate reductase mRNA levels could not be ascertained. These results suggested that post-translational control mechanisms were involved in nitrate reductase regulation during in vitro tobacco callus differentiation. / Thesis-(M.Sc.)-University of Natal, Durban, 1993.