Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

Abstract

MATERIALS AND METHODSIn the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: a-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.Changes in the activities of alanine: a-ketoglutarate aminotransferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.13) have also been measured, in cotyledons as well as seedcoats. On a fresh weight basis, the highest activities of asparaginase and both aminotransferases developed in the seedcoats, whereas the highest activities of the remaining enzymes developed in the cotyledons.The data indicate that the amide groups of imported asparagine and glutamine are metabolized differently, largely by asparaginase and glutamate synthase, respectively. The NH4' released by the action of asparaginase is evidently reassimilated in cotyledon cells by the joint action of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. The data emphasize the central importance of a-ketoglutarate-glutamate cycling in the redistribution of amino groups associated with the net synthesis of amino acids and reserve proteins. PLANT MATERIAL AND PREPARATION OF EXTRACTSPlants of tall (cv. Telephone) and dwarf (cv. Melbourne Market) varieties of garden pea, P. sativum L., were grown, respectively, in the open garden or in pots of garden soil. Flowers were tagged at full blossom and pods then were removed at intervals throughout development, which occurred in spring (October, November) for cv. Melbourne Market and from November to mid-December (1979) for cv. Telephone.The procedures for extraction of separated seedcoat and cotyledon samples were as described (17, 18), with the sample size increased to four or five seeds. The extraction medium consisted of 25 mm K+-phosphate (pH 7.5), 0.5 mm 2-mercaptoethanol and 0.03% (v/v) Triton X-100. After centrifugation (17), duplicate aliquots were removed for treatment with ethanol, for the determination of protein content (ethanol-insoluble) and a-amino group content (ethanol-soluble) as previously described (16, 17).Measured portions of each clarified extract (1.0-1.5 ml) were passed through columns of Sephadex G-25 (0.9 x 25 cm) equilibrated with extraction medium to obtain enzyme solutions free of NH4, and amino acids. Complete removal of ethanol-soluble, ninhydrin-positive metabolites from the excluded fractions was confirmed by analysis (17).A number of recent studies have attempted to elucidate the nutritive and regulatory influences that the living seedcoats exert upon the embryo during its development (4,(17)(18)(19). Lacking the phenolic compounds that hinder the extraction of proteins from the seedcoats of many other species, developing pea seeds (Pisum sativum L.) offer an ideal system in wh...

15N and 13C NMR Determination of Allantoin Metabolism in Developing Soybean Cotyledons

“…In peas, the seed coat may play an active role in the nutrition of the cotyledons (8,9). For example, high levels of asparaginase are present within the seed coat suggesting that asparagne is degraded within the seed coat (9). For soybeans, the presence of higher levels of GS and higher rates of allantoin degradation in the seed coat than in the cotyledons (Table V) …”

“…Nitrogen leaving the seed coat exits primarily as glutamine, asparagine, and NH4' (10); ureides do not exit from the seed coat. In peas, the seed coat may play an active role in the nutrition of the cotyledons (8,9). For example, high levels of asparaginase are present within the seed coat suggesting that asparagne is degraded within the seed coat (9).…”

Concentrations of Sucrose and Nitrogenous Compounds in the Apoplast of Developing Soybean Seed Coats and Embryos

“…Specifically, the data presented on apoplastic solute concentrations provide information as to what solutes get into the seed, and what kind of processing these solutes undergo between the site of unloading and the site of uptake by the embryo. As indicated in Table I (14), the high concentration of GOGAT activity in the nucellus suggested that solutes may pass through the symplasm ofthis tissue layer. The transport properties ofthis strategically located tissue deserve further study.…”

ADP-Glucose Pyrophosphorylase-Deficient Pea Embryos Reveal Specific Transcriptional and Metabolic Changes of Carbon-Nitrogen Metabolism and Stress Responses

“…Three up-regulated genes were involved in aromatic amino acid metabolism, 3-dehydroquinate synthase, indole-3-glycerol phosphate synthase, and fumarylacetoacetase, the latter of which catalyzes Phe degradation. In addition, asparaginase, putatively involved in N mobilization (Murray and Kennedy, 1980), was transcriptionally up-regulated at all four stages. Its product Asp can be converted to Glu via Asp transaminase, which was also up-regulated.…”