Robert T. Leonard

Contamination of Ammonium-Based Nutrient Solutions by Nitrifying Organisms and the Conversion of Ammonium to Nitrate

Conversion of ammonium to nitrate and contamination by nitrifying organisms are often assumed not to be significant in ammonium-based nutrient solutions. To assess this assumption, maize (Zea mays) and pea (Pisum sativum) were grown under greenhouse conditions in aeroponic, hydroponic, and sand-culture systems containing 2 mM ammonium chloride as the sole nitrogen source and evaluated for the activity of contaminating nitrifying organisms. In all three culture systems, root colonization by nitrifying organisms was detected within 5 d, and nitrate was detected in the nutrient solution within 10 d after seedling transfer. In sand culture, solution nitrate concentration reached 0.35 mM by the end of the 17-d experiment. Consistent with the microbial ammonium oxidation sequence, nitrite was detected earlier than nitrate and remained at lower levels throughout the experiment. Nitrate was found in significant quantities in root and shoot tissues from seedlings grown in ammonium-based nutrient solutions in all of the solution culture systems. Maize seedlings grown in an ammonium-based hydroponic system contained nitrate concentrations at 40% of that found in plants grown in nitrate-based solution. Determination of nitrate (or nitrite) levels in the nutrient solution was the weakest indicator of the activity of nitrifying organisms. A bioassay for the presence of nitrifying organisms in combination with tissue analysis for nitrate was a better indicator of microbial conversion of ammonium to nitrate in nutrient solution culture. The results have implications for the use of ammonium-based nutrient solutions to obtain plants suitable for research on induction of nitrate uptake and reduction or for research using solution culture to compare ammonium versus nitrate fertilization.

Isolation of plasma membrane ofPhytophthora megasperma f. sp.glycinea and some properties of the associated ATPase

Abstract Plasma membrane vesicles were isolated from Phytophthora megasperma f. sp. glycinea using conventional methods of mechanical disruption followed by differential and density gradient centrifugation. The validity of presumed biochemical markers was confirmed using electron microscopy and the phosphotungstic acid-chromic acid staining procedure, which was judged to be specific for plasma membrane when performed under suitable conditions. The plasma membrane fraction showed a peak equilibrium density of 1.14 g/ml and was identified by its vanadate-sensitive Mg 2+ -dependent ATPase with an optimum temperature of 42°C and a pH optimum of 6.0 to 6.5. The activity was weakly stimulated by K + and strongly inhibited by Ca 2+ . The enzyme showed a marked specificity for ATP as a substrate compared to other nucleoside mono-, di-, and triphosphate substrates or other general phosphatase substrates. The divalent cation requirement could be met equally well by Mg 2+ and Co 2+ and, to a lesser extent, by Mn 2+ , but not by Ni 2+ , Ba 2+ , Zn 2+ , Sr 2+ , Ca 2+ , Hg 2+ , Cu 2+ , or Fe 2+ (in decreasing order of preference). Contamination by intact mitochondria (density 1.21 g/ml) or mitochondrial fragments (density 1.16 g/ml) was minimal and could be monitored by measuring cytochrome c oxidase or oligomycin-sensitive pH 8.5 ATPase.

Phenol-acetic acid-urea polyacrylamide gel electrophoresis of membrane proteins☆

A phenol-acetic acid-urea polyacrylamide gel electrophoretic system (PAU-PAGE) was simplified by adaptation to a slab gel format, allowing the simultaneous comparison of up to 12 samples. The system fractionated most proteins according to molecular mass, although chemical reduction was required since certain proteins (e.g., bovine serum albumin) showed reduction-dependent shifts in mobility. Sodium dodecyl sulfate-PAGE of partially purified membrane proteins can be adversely affected by protein aggregation and proteolysis. PAU-PAGE, which solubilized aqueous insoluble proteins and rapidly inactivated proteases, was useful for assessing the polypeptide composition of plasma membrane preparations.

An amino-acid-grown maize cell line for use in investigating nitrate assimilation

SummaryStudies on uptake and assimilation of nitrate in plants are confounded by differences in cell function associated with anatomical features of roots as well as by problems inherent with growing plants without nitrate. To circumvent these problems, a Zea mays L. embryo cell line was grown in suspension culture using an amino-acid-based medium consisting of a Murashige and Skoog medium in which ammonium and nitrate were replaced by aspartic acid (100 mg/l), glycine (100 mg/l), arginine (150 mg/l), and glutamine (1 g/l). The growth, cellular characteristics, and physical appearance of the amino-acid-grown cells were similar to cells grown in the presence of nitrate. The amino-acid-grown cells exhibited the expected induction pattern and inhibitor sensitivity of nitrate uptake. This cell line should facilitate research on the induction of nitrate uptake and the regulation of nitrate assimilation into proteins.