Development of Pyrimidine-metabolizing Enzymes in Cotyledons of Germinating Peas

Abstract

Mechanisms controlling conversion of orotic acid-6-'4C to uridine-5'-phosphate in cotyledons of germinating Alaska peas (Pisum sativum L.) were investigated. The content of 5-phosphoribosyl-l-pyrophosphate was very low in dry seeds, increased to a maximum after about 12 hours of imbibition, and then rapidly declined. Orotidine-5'-phosphate pyrophosphor. ylase and orotidine-5'-phosphate decarboxylase activities more than doubled during the first 24 hours of germination and then also decreased. These results do not account for the continuous increases of orotate anabolism in such cotyledons as we observed previously. The initial increases in activities of these two enzymes were unaffected by cycloheximide, while the subsequent decreases were less rapid in the presence of this inhibitor. Activities of cotyledonary cytidine deaminase and uridine hydrolase also increased during imbibition, but the activity of only the latter showed a decrease after imbibition was completed. Cycloheximide inhibited the initial rapid increase in uridine hydrolase activity but had little effect on its subsequent decline. Cycloheximide had only slight inhibitory effects on the development of cytidine deaminase activity during the first 62 hours. The evidence suggests that uridine hydrolase might be synthesized de novo during the first few days of germination, but that the other three enzymes might not be.We previously observed that "C-labeled orotic acid and uridine were converted into various pyrimidine nucleotides and RNA in cotyledons of germinating Alaska peas (15). This incorporation was more extensive in seeds germinated about 1 week than in those germinated for lesser times. It was suggested that the increasing capacity of the seeds to convert orotate into RNA might be due to an increase of OMP-pyrophosphorylase activity or of PRPP, a necessary substrate for this enzyme. Decarboxylation of OMP appeared to be nonlimiting at all germination stages, since no "C-labeled OMP was ever detected in the cotyledons. On the contrary, orotate-"C accumulated in seeds which only weakly converted it into RNA. The present report describes results indicating that both OMP pyrophosphorylase and OMP decarboxylase become increasingly active in pea cotyledons during the first 24 to 36 hr of germination, but their activities then decrease. The decreases but not the increases in enzyme activities are inhibited by CH, a known inhibitor of protein synthesis. The effects of CH on protein synthesis and on the development of cytidine deaminase and uridine hydrolase, two enzymes suspected to be involved in the metabolism of RNA stored in the cotyledons, are also described. MATERIALS AND METHODSGerminaton. Pea (Pisum sativiuim L., cv. Alaska) seeds were germinated under conditions designed to minimize bacterial contamination. Seeds were shaken in 1.0% (w/v) NaOCl for 3 min and rinsed extensively in sterile water. Twenty seeds were then spread over the surface of three layers of Whatman No. 1 filter paper in 9-cm Petri dishes. The Petri dishes and filt...

Pyrimidine Metabolism in Lemna minor

“…Bleaching by Cdr is fully prevented by thymidine (Tdr), cytidine (Cr), or uridine (Ur), but The isolation of purine and pyrimidine auxotrophs in higher plants, with the possible exception of Arabidopsis (17), has not proven possible. This has restricted description of existing metabolic pathways to selective confirmation of certain enzyme activities and to the use of only certain types of tissues (1,4,7,20 (Fig. Ib).…”

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

“…Preliminary results with another of the pyrimidine pathway enzymes, dihydroorotase, suggest it also follows the same inhibitor stimulation pattern in cowpea hypocotyls as does ATCase (Johnson, unpublished). Also, two other pathway enzymes, orotidine-5'-P pyrophosphorylase and orotidine-5'-P decarboxylase, increase during germination in pea in the presence of CH (29). Thus several enzymes involved in the pyrimidine pathway may be regulated in some tissues by a mechanism that responds abnormally to inhibitor treatment.…”

“…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. Reported here are unusual stimulatory effects of certain inhibitors on extractable ATCase activity from etiolated cowpea hypocotyls.…”

“…Control hypocotyls were immersed in H20 and treated comparably. The (4,7,9,29). To test that hypothesis we excised cowpea hypocotyls at the root-hypocotyl juncture 6 days after planting and treated them with either 96 ,ug/ml AMD, 100 l.g/ml CH, or 2.5 mg/ml CHL solutions (Table I, experiment 1).…”

Properties of Pea Seedling Uracil Phosphoribosyltransferase and Its Distribution in Other Plants

“…There is also evidence for uridine phosphorylase in pea, wheat, barley, and Lathyrus tingitanus (13,25), so there are apparently two mechanisms for uracil salvage in plants. However, UPRT appears to be considerably more active than the phosphorylase, and the latter enzyme was undetectable in pea cotyledons (20) and in mung bean seedlings (1).…”