Isolation of cDNAs encoding two purine biosynthetic enzymes of soybean and expression of the corresponding transcripts in roots and root nodules (original) (raw)
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Enzymes of Amide and Ureide Biogenesis in Developing Soybean Nodules
Plant Physiology, 1982
Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period II to 23 days after inoculation with Rhizobiumjaponicum (USDA 311b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5-phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaler increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase. The concentration of asparagine in the plant fraction decreased at the same time as the observed decrease in asparagine synthetase activity. This was followed by a recovery in plant fraction levels of asparagine in the presence of a continuing fall in the glutamine concentration and continued low asparagine synthetase activity. The data presented are consistent with initial assimilation of ammonia into glutamine and aspartate, which are metabolized by an elevation of endogenous purine biosynthetic enzymes, and then, by the induction of a specific group of purine oxidative enzymes, directed to allantoic acid production. The mechanism of the assimilation of fixed N into the ureides, allantoin and allantoic acid, still remains to be clearly elucidated in those leguminous plants which use these compounds fot the 'Supported by a grant from the United States Department of Agriculture, Science and Education Administration Competitive Research Grants Office (Grant No. 5901-0410-9-0231 to D. G. B. and No.
Phosphoserine Aminotransferase in Soybean Root Nodules
Plant Physiology, 1986
ABSTRACI Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine-pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules. ' Supported in part by a grant from the United States Department of Agriculture, Science and Education Administration Competitive Research Grants Office (Grant No. 81-CRCR-1-0672 to D. G. B.). Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 996 1. 2 Recipient of a United Nations Fellowship from the Organisation for Economic Cooperation and Development Cooperative Project on Food Production and Preservation.
PLANT PHYSIOLOGY, 1982
Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine mar L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N2 per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N2 per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate www.plantphysiol.org on May 25, 2020 -Published by Downloaded from
Plant Physiology
Gln synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to yield Gln. In higher plants GS is an octameric enzyme and the subunits are encoded by members of a small multigene family. In soybeans (Glycine max), following the onset of N2 fixation there is a dramatic increase in GS activity in the root nodules. GS activity staining of native polyacrylamide gels containing nodule and root extracts showed a common band of activity (GSrs). The nodules also contained a slower-migrating, broad band of enzyme activity (GSns). The GSns activity band is a complex of many isozymes made up of different proportions of two kinds of GS subunits: GSr and GSn. Root nodules formed following inoculation with an Nif- strain of Bradyrhizobium japonicum showed the presence of GS isoenzymes (GSns1) with low enzyme activity, which migrated more slowly than GSns. Gsns1 is most likely made up predominantly of GSn subunits. Our data suggest that, whereas the class I GS genes encod...
Plant Science, 1997
A 3.5 kb promoter fragment fused to the reporter i-glucuronidase gene (gus) had previously been shown to contain separate regulatory elements controlling the ammonia-stimulated and organ-specific transcription of GS15, a soybean gene encoding root and root nodule cytosolic glutamine synthetase (GS). In order to determine if the regulatory elements conferring the ammonia-regulated and the organ-specific expression are conserved in different plant species, a GS15 promoter-gus (pGS15GUS) construct was introduced via Agrobacterium-mediated transformation both in tobacco and alfalfa. Histochemical localisation of GUS activity revealed that, in both heterologous systems, the cytosolic GS gene was expressed in anthers, theca and pollen at a late stage of flower development. Strong GUS staining was also visible in transgenic alfalfa pulvini and petioles. pGS15GUS was also found to be expressed in roots, however, treatment with ammonia did not increase the expression of the reporter gene either in tobacco or alfalfa. In mutant nodules of alfalfa formed by two different Fix − strains of Rhizobium meliloti both GUS staining and GUS activity were similar to the Rhizobium wild-type infected nodules indicating that GS15 expression in alfalfa root nodules does not depend on the production of ammonia coming from symbiotically fixed nitrogen. The results are discussed in relation to the possible role of cytosolic GS in different organs of legumes and other plant species.
Plant Molecular Biology, 1994
Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia colipurE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO2 and is dispensable in the presence of high CO2 concentrations. Because the expression of the mothbean Pure cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO2-binding activity.
Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes
Plant Journal, 1994
Glycinamide ribonucleotide (GAR) synthetase, GAR transformylaes and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cONAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherlchia coil mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coliexpreesing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.
Archives of Biochemistry and Biophysics, 1984
Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de nova purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent M, of 8 X 106, but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required M$+, and PRPPM$-was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the V, with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine,
5—Phosphoribosylpyrophosphate Amidotransferase From Soybean Nodules
Springer eBooks, 1984
Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de nova purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent M, of 8 X 106, but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and Km values were 18 and 0.4 mM for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required M$+, and PRPPM$-was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the V, with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine,