Identification and characterization of an mRNA encoding a proline-rich protein that rapidly declines in abundance in the tips of harvested asparagus spears.

We previously isolated a cDNA clone, pTIP13, whose homologous mRNA rapidly declined in abundance in the tips of harvested asparagus (Asparagus officinalis L.) spears [King and Davies (1992) Plant Physiol. 100: 1661]. In order to identify factors regulating the postharvest deterioration of asparagus, we have now sequenced the pTIP13 cDNA, derived the encoded amino acid sequence and determined the cellular location of pTIP13 mRNA by in situ hybridization. pTIP13 encodes a derived protein that is rich in proline (22.3%), but also has a high content of lysine (15.2%) and threonine (14.1%). The proline residues are located in motifs at the amino-terminal region of the protein. The carboxyl-terminal region of the derived protein has a high leucine content and shares > 64% amino acid identity with derived proteins identified from cDNA clones to cell wall protein precursor mRNAs obtained from soybean hypocotyls, alfalfa roots, and tomato fruit. Genomic Southern analysis suggests that pTIP13 is encoded by a single-copy gene in asparagus. pTIP13 mRNA was localized to specific cell types in the young bracts of the asparagus spear tip. The results provide new information on the complexity of tissue responses in the tips of asparagus spears following harvest.

Evolutionary analysis of aspartate aminotransferases.

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family.

Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity.

The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors. Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots. The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained. The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region. The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2 homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.

Repression of the L-asparaginase gene during nodule development in Lupinus angustifolius.

Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased. This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development. Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules. This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds. The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site. Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments. Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein. We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

Cloning and characterization of a cDNA encoding aspartate aminotransferase-P1 from Lupinus angustifolius root tips.

A root tip cDNA library, constructed in the lambda Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P1 from Lupinus angustifolius L. var Uniharvest. One 1452-base pair clone was isolated. The encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences. The clone was converted to the phagemid form and expressed in Escherichia coli. The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies. The complete aspartate aminotransferase-P1 cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCS6, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation). Complementation of the mutation was achieved using this construct.

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.

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.

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.

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase.

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.

Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase.

Activities and properties of the ammonium assimilation enzymes NADP+-dependent glutamate dehydrogenase (GDH), glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined in batch and continuous cultures of Candida albicans. NADP+-dependent GDH activity showed allosteric kinetics, with an S0.5 for 2-oxoglutarate of 7.5 mM and an apparent Km for ammonium of 5.0 mM. GOGAT activity was affected by the buffer used for extraction and assay, but in phosphate buffer, kinetics were hyperbolic, yielding Km values for glutamine of 750 microM and for 2-oxoglutarate of 65 microM. The enzymes GOGAT and NADP+-dependent GDH were also assayed in batch cultures of Saccharomyces cerevisiae and three other pathogenic Candida spp.: Candida tropicalis, Candida pseudotropicalis and Candida parapsilosis. Evidence is presented that GS/GOGAT is a major pathway for ammonium assimilation in Candida albicans and that this pathway is also significant in other Candida species.

Characterization of the peribacteroid membrane ATPase of lupin root nodules.

Peribacteroid membranes can be isolated in essentially pure form from 20-day lupin root nodules by osmotic shock of the purified membrane enclosed bacteroids. The ATPase (EC 3.6.1.3) associated with this membrane has an acid pH optimum (5.25) and is specific for ATP (Mg-ATP Km = 0.16 mM). The enzyme activity requires magnesium or manganese ions, is slightly stimulated by the cations potassium and rubidium, and is inhibited by vanadate, diethylstilbestrol, N,N'-dicyclohexylcarbodiimide, fluoride, molybdate, and calcium. Molybdate and fluoride sensitivity do not in this case indicate the presence of significant nonspecific phosphatase activity. The ATPase is not inhibited by oligomycin, azide, or the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some respects the lupin peribacteroid membrane ATPase appears to differ from the plasma membrane ATPase of other plants.

Stereochemical aspects of the biosynthesis of the epimeric cyanogenic glucosides dhurrin and taxiphyllin.

Dhurrin, I ((S)-p-hydroxymandelonitrile-beta-D-glucopyranoside), and taxiphyllin, II (the (R) epimer), occur in the genera Sorghum and Taxus, respectively. Both derive biosynthetically from L-tyrosine via the hydroxylation of p-hydroxyphenylacetonitrile, III. (3R)- and (3S)-L-[3-3H1]tyrosine, prepared by enzymic hydroxylation of the corresponding phenylalanines, were fed separately to shoots from sorghum seedlings (Sorghum bicolor (Linn) Moench) and cuttings from Japanese Yew (Taxus cuspidata Sieb. and Zucc.) and the appropriate cyanogenic glycoside was isolated (I or II). The fraction of the 3H conserved in I and II was calculated from both parallel feeding and 3H:14C double labeling experiments. The results for II were the reverse of I. Both hydroxylations of III, which give rise to the enantiomeric products (S)- and (R)-p-hydroxymandelonitrile, occurred with retention of configuration. The retention mode is characteristic of hydroxylations at aliphatic carbons catalyzed by mixed function oxidases.