Paul H. S. Reynolds

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

A system for tissue-specific copper-controllable gene expression in transgenic plants : nodule-specific antisense of aspartate aminotransferase-P2

A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeastace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulatedLotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue ‘wild-type tops/transgenic roots’. After addition of copper ions to the plant nutrient solution, β-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when theace1 gene was placed under control of thenod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenicLotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotranferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.

The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus)

An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.

Ureide biogenesis in leguminous plants

Abstract Allantion and allantoic acid are the predominant forms of organic nitrogen produced by N-fixing nodules of some important legume species. Only recently has the significance and biosynthetic origin of these substances in leguminous plants been realized.

Enzymes of nitrogen metabolism in legume nodules: Partial purification and properties of the aspartate aminotransferases from lupine nodules

Abstract The two aspartate aminotransferase isoenzymes, AAT-P1 and AAT-P2, found in the plant cytosol fraction of lupine nodules have been separated and partially purified. Both isoenzymes showed a broad pH optimum between 7.0 and 9.5 and demonstrated high substrate specificity. Molecular weights determined by gel filtration chromatography were 105,000 and 96,000 for AAT-P1 and AAT-P2, respectively. AAT-P1 demonstrated a subunit molecular weight of 47,000 and AAT-P2, 45,000. Both isoenzymes showed oxaloacetate substrate inhibition and gave similar Km values for aspartate (2.2 and 2.6 m m ) and 2-oxoglutarate (0.26 and 0.2 m m ) for AAT-P1 and AAT-P2, respectively. However, AAT-P2 showed a fivefold lower Km for oxaloacetate (0.02 m m ) and a 1.8-fold lower Km for glutamate (12 m m ) than did AAT-P1 for these substrates. The parallel nature of the 1 V vs 1 [ S ] plots together with the product inhibition kinetics were consistent with a ping-pong-bi-bi mechanism of action. The results are discussed in terms of the possible physiological significance of these plant aspartate aminotransferases in ammonia assimilation in lupine nodules.

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Abstract Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini . Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini . The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.

5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: Kinetic and regulatory properties

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 novo 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 Mr of 8 X 10(6), 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 Mg2+, and PRPPMg3- was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the Vm 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, PPi, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.

L-asparaginase from developing seeds of Lupinus arboreus

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.

Intra-cellular localization of the biosynthetic pathway to flavour precursors in onion

Abstract The contents of protoplasts from leaves of sprouted onions (unlabelled and labelled with 35S) were separated into chloroplasts, mitochondria and cytoplasm or sucrose density gradients. 35S labelled γ-glutamyl cysteine and glutathione were found in the chloroplasts, and labelled glutathione, γ-glutamyl peptides and flavour precursors were found in the cytoplasm. γ-Glutamyl cysteine synthethase was shown to be localized in the chloroplasts, whilst γ- glutamyl transpeptidase was found in the cytoplasm. The data suggest that the biosynthesis of glutathione occurs in the chloroplasts and subsequent metabolism from glutathione, via the γ-glutamyl peptides to flavour precursors is cytoplasmic.

Molecular cloning of the gene encoding developing seed L-asparaginase from Lupinus angustifolius

A genomic sequence encoding Lupinus angustifolius L-asparaginase has been obtained, and is the first report of this gene from a plant source. The 3.2 kb of DNA sequenced contains a 1136 bp 5′ flanking sequence, four exons and three introns. Intron-exon borders were mapped by comparing the genomic sequence with that of a L. arboreus cDNA. Primer extension analysis revealed transcription start sites 16 bp and 13 bp 5′ of the initiating ATG for L. angustifolius and L. arboreus, respectively. The 5′ flanking region contained sequences associated with seed-specific expression.