Kevin J. F. Farnden

Cyanide metabolism in higher plants: V. The formation of asparagine from β-cyanoalanine☆

β-Cyanoalanine hydrolase, an enzyme which catalyzes the formation of asparagine from β-cyanoalanine, has been isolated from the soluble fraction of the cotyledons and shoots of 11-day-old etiolated blue lupin seedlings. The enzyme can be assayed radio-chemically by measuring the incorporation of label from β-[4-14C]cyano-l-alanine into asparagine, or colorimetrically by estimating the ammonia produced by Nesslerization after hydrolysis of the asparagine formed during the incubation. The enzyme has been purified some 250–300 fold, though not to homogeneity, with an overall yield of 15%. β-Cyanoalanine hydrolase has a pH optimum of about 8.5 and a molecular weight of 400,000–500,000 estimated from its elution volume on Sepharose 6B. A Km of 2.0 mm for β-cyanoalanine was determined. The blue lupin β-cyanoalanine hydrolase was found to be highly specific for β-cyanoalanine. This enzyme was distinct from other plant, bacterial and fungal nitrilases, asparaginases or glutaminases and plant hydroxynitrile lyases

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.

Purification and properties of asparaginase from Lupinus arboreus and Lupinus angustifolius.

Abstract l -Asparaginase (EC 3.5.1.1.) activity has been detected in crude extracts of Lupinus arboreus young leaves, root tips, flower buds, and developing seeds. The enzyme was also present in Lupinus angustifolius root tips, developing nodules, and developing seeds. The asparaginase from each of these tissues had the same electrophoretic mobility on polyacrylamide gels and a K m of 6–8 m m for asparagine. In extracts other than those of the developing seeds, asparaginase activity was dependent upon the inclusion of K + ion and a sulfhydryl protectant in the extraction buffer. No asparaginase activity was detected in mature leaves, in the plant fraction of nodules that were fixing nitrogen, nor in root tissue further than 1.5 cm from the root tip. Asparaginase has been purified 326- and 230-fold from L. arboreus and L. angustifolius developing seeds, respectively. A molecular weight of 75,000 was obtained by gel filtration. An apparent K m of 6.6 and 7.0 m m for asparagine was determined for the purified L. arboreus and L. angustifolius asparaginases, respectively. Of the amides, nitriles, and hydroxamates examined, the L. arboreus enzyme hydrolyzed only l -asparagine and dl -aspartyl hydroxamate. This same enzyme was inhibited by d -asparagine, 5-diazo-4-oxo- l -norvaline, dl -aspartyl hydroxamate, d -and l -aspartate, 3-cyano- l -alanine, glycine, and cysteine. Glutamine, glutamine analogs, and a number of other amino acids, amides and amines did not inhibit the L. arboreus asparaginase.

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.

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers.

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.

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Abstract Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 m m ; l -glutamate, 4.0–4.2 m m ; ammonium, 0.16 m m . Using an hydroxamate-forming biosynthetic reaction the K m ATP is 1.1 m m but the K m for l -glutamate is not altered. The effect of pH on the K m for ammonium indicates that NH 3 rather than NH 4 + may be the true substrate. At 10 m m Mg 2+ , the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg 2+ concentrations produce progressively more acidic optima while lower Mg 2+ concentrations raise the pH optimum. The rate-response curve for Mg 2+ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l -Asp ( K i , 1.4 m m ) and less markedly by l -Gln and l -Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.

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.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the -1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in -1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than -405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least -640 bp of the AS promoter but not those with -523 bp or smaller promoter fragments. Fusion of the -640 to -523 bp region to a -381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.