Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used '4C-aspartate, which was then separated from the "4C-ureidosuccinate product by Dowex 50 (H+ form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivlity cochromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5'-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.Aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) catalyzes the first reaction unique to pyrimidine biosynthesis: aspartic acid + carbamyl phosphate -* carbamyl aspartate (ureidosuccinate) + P,. This enzyme is regulated by feedback inhibition in several microorganisms (22). The pathway of synthesis of pyrimidine nucleotides in higher plants appears similar to that reported for microorganisms (26). Aspartate transcarbamylase from higher plants is a potential site for feedback regulation of the pathway, as it is inhibited strongly by UMP and somewhat by certain other pyrimidine nucleotides (19,20,28,29 cant increase in the activity of this enzyme should occur following 2, 4-D treatment. MATERIALS AND METHODSPlant Material. Etiolated cowpea (Vigna unguiculata L. Walp cv. Early Ramshorn) seedlings were grown in the dark at 29 to 30 C and 85% relative humidity. Seeds with unbroken seedcoats (15) were surface-sterilized 2 to 3 min in 0.5% NaOCI and planted in trays of moist vermiculite.Five days after planting, seedlings were sprayed to run-off with a 2,4-D solution (1 mg/ml) containing 4% ethanol. Controls received a comparable 4% ethanol spray. Hypocotyls were harvested at 12-hr intervals and stored at -18 C until extracted.Tissue Extraction. Frozen tissues were ground with a cold mortar and pestle in cold (3 C) 0.1 M ethanolamine-HCl buffer, pH 10.0. Five or six hypocotyls were ground per treatment, in 2 ml of buffer per g fresh weight. Extracts were centrifuged at 37,000g and 3 C for 15 min. Aliquots of the supernatant fluid were frozen for later determination of RNA, DNA, and soluble protein. Another portion was dialyzed for 16 to 20 hr in cold 0.05 M ethanolamine-HCl buffer, pH 10.0, for use in enzyme assays.Aspartate Transcarbamylase Assay. Four test tubes containing 0.5 ml of dialyzed enzyme and 0.25 ml of 0.05 M ethanolamine-HCl (pH 10.0) buffer were prepared for each treatment and stored at 3 C until 3 to 8 min before assay, when the tubes were shaken in a water bath at 37 C. The ...

Mycelial discs of Rhizoctonia solani were treated with the oxathiin systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) at 10−4 M to determine the effect upon fungal plasma membranes. Electrical conductance was used as a preliminary monitor for cell leakage. Carboxin treatment consistently resulted in the release of ionic materials. Potassium ions, organic acids, and phosphate-containing materials were exuded from cells treated with carboxin. Phosphate losses were sufficient to retard, but not to stop, further growth of the organism after removal of the fungicide and placement of the mycelium on a growth medium lacking phosphate. Starvation of mycelial discs in deionized water markedly reduced respiratory metabolism and carboxin treatment after starvation did not result in cell leakage, thus indicating that membrane damage did not occur. Nystatin, a known disrupter of membranes, did cause leakage from starved cells. Inasmuch as carboxin is known to inhibit respiratory metabolism, organic acids and phosphate-containing materials may accumulate in treated cells and then be exuded as a secondary effect.