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

Abstract

Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Waip.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cowpea hypocotyls with cydobeximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. 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 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. Control hypocotyls were immersed in H20 and treated comparably. The five seedlings of each set were then placed with their bases in a test tube (18 x 150 mm) containing 5 ml of 20 mm K-phosphate buffer, pH 5.6. Test tube tops were sealed with Parafilm around the emerging epicotyls, and the tubes were returned to the dark at 29 to 30 C. Five seedlings were used per sample, and duplicate samples were used for each treatment. All operations were in dim light. At harvest, the basal cm of each hypocotyl was discarded, and the next 11 cm were weighed, coarsely diced, and frozen until extracted.Hypocotyl Extraction. Hypocotyl sections were homogenized for 15 sec with a Willems Polytron PT 20 ST, with the speed control at five. Homogenization was in 2 ml of cold 0.1 M ethanolamine-HCl buffei, pH 10 for cowpea and pH 9.5 for soybean, per g fresh weight in 50-ml centrifuge tubes kept in ice H20. Samples were centrifuged 15 min at 37,000g and 3 C.Aliquots of the supernatant were frozen for later analysis of RNA and buffer-soluble protein, and the remainder was dialyzed for 16