Asparate Transcarbamylase Activity in Etiolated Cowpea Hypocotyls Treated with 2,4-Dichlorophenoxyacetic Acid

Abstract

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 ...

Effects of Inhibitors of RNA and Protein Synthesis on Aspartate Transcarbamylase Activity in Etiolated Plant Tissue

“…In higher plants, ATCase activity is elevated in young tissue and decreases as tissue matures (14,24,30). Declining activity of some enzymes in eukaryotic organisms may be mediated through enzyme degradation or inactivation, processes frequently blocked by inhibitors of RNA or protein synthesis (4,7,29 consistent with such regulation (13) and prompted this work.…”

“…cv. Wayne) were grown in darkness in trays of moist vermiculite at 29 to 30 C and 85 to 90% relative humidity as described (14).…”

“…In higher plants, ATCase activity is elevated in young tissue and decreases as tissue matures (14,24,30 (14).…”

“…In contrast erythromycin had no effect, and D-threo-chloramphenicol (CHL) ATCase2 (25). In higher plants, ATCase involvement in pyrimidine regulation is not established, although it likely has a regulatory role because the enzyme from several plant species is UMPsensitive in vitro (1,14,23,26,33). ATCase activity increases during times of enhanced nucleic acid synthesis, including seed germination (21), response to 2,4-D (14), and after virus infection (24).…”

“…In higher plants, ATCase involvement in pyrimidine regulation is not established, although it likely has a regulatory role because the enzyme from several plant species is UMPsensitive in vitro (1,14,23,26,33). ATCase activity increases during times of enhanced nucleic acid synthesis, including seed germination (21), response to 2,4-D (14), and after virus infection (24).In higher plants, ATCase activity is elevated in young tissue and decreases as tissue matures (14,24,30 (14).Inhibitors were applied to seedlings uprooted 7 to 8 days after planting, after roots were excised underwater at the hypocotylroot juncture. Hypocotyls were immersed in sets of five into the proper solutions, placed in a vacuum desiccator, and a vacuum was drawn for 3 min, and then slowly released.…”