Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein.
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The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds. (+info)
A gene for the delta9 desaturase specific to stearoyl-ACP (acyl carrier protein) was identified from yellow lupine (Lupinus luteus) cDNA and genomic libraries through the differential display method. The desaturase transcript appears in plants infected with Bradyrhizobium sp. (Lupinus) as revealed by Northern hybridization, RT-PCR and expression of beta-glucuronidase under the desaturase promoter. A small amount of desaturase transcript was also detected in uninfected plants, which suggests that the gene does not belong to the strict nodule-specific sequences. The desaturase provides unsaturated fatty acids for additional cell membrane synthesis. During nodule and symbiosome development a peribacteroid membrane is formed and the requirement for membrane surface increases, thus the level of desaturase expression is also higher. Transgenic plants of Nicotiana tabacum with overexpression of the full-length lupine stearoyl-ACP desaturase sequence were obtained. They revealed higher content of unsaturated fatty acids (especially oleic acid) in comparison with control plants. (+info)
Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L. cv Estoril.
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Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT- 2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism. (+info)
Folliculogenesis and ovarian expression of mRNA encoding aromatase in anoestrous sheep after 5 days of glucose or glucosamine infusion or supplementary lupin feeding.
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Improved nutrition increases ovulation rate in sheep and there is evidence that intra-ovarian pathways mediate responses to nutrition. An experiment was conducted to examine the effect of dietary energy on folliculogenesis. Anoestrous Merino ewes were fed a diet of wheat straw alone (control, n = 5), or wheat straw supplemented with lupins (500 g day(-1), n = 5). Other ewes were fed wheat straw and infused with glucose (50 mmol h(-1), n = 5) or with glucosamine (3.5 mmol h(-1), n = 5). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before sponge removal. At sponge removal, the ewes were injected with a regimen of GnRH pulses (500 ng every 4 h from 0 to 12 h; 250 ng every 2 h from 14 to 24 h; and 200 ng every 1 h from 25 to 36 h) to simulate normal follicular development. Thirty-six hours after sponge removal, the animals were killed and the ovaries were collected and stored at -80 degrees C. The ovaries were sectioned serially every 10 microm. Every 20th section was stained (to estimate number and diameter of follicles) and every 17-19th section was probed by in situ hybridization for P(450) aromatase. Data were analysed using ANOVA and chi-squared tests. There was an effect of treatment (P < 0.05) on the number of follicles 2-3, 3-4 and 6-7 mm in diameter. Aromatase-positive follicles (1.6-7.9 mm) were detected in 31 follicles from 15 ewes across all four groups. In ten animals, the largest follicle was aromatase-positive. The diameters of aromatase-positive follicles were larger (P = 0.004) in lupin fed compared with glucose-infused ewes (4.9 +/- 0.5, 3.6 +/- 0.7, 5.3 +/- 0.5 and 4.2 +/- 0.5 mm for control, glucose-infused, lupin-fed and glucosamine-infused groups, respectively). Treatment did not affect the plasma concentration of FSH when compared with controls, indicating that the energy supplements were modifying recruited (2-3 mm and 3-4 mm) and selected follicles (> 6 mm) directly. In conclusion, dietary energy can directly stimulate folliculogenesis in recruited and selected follicles, and this effect may be mediated by changes in systemic leptin concentrations and the hexosamine energy-sensing pathway in the follicle. (+info)
Metabolism of homocysteine-thiolactone in plants.
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Editing of the amino acid homocysteine (Hcy) by certain aminoacyl-tRNA synthetases results in the formation of an intramolecular thioester, Hcy-thiolactone. Here we show that the plant yellow lupin, Lupinus luteus, has the ability to synthesize Hcy-thiolactone. The inhibition of methylation of Hcy to methionine by the anitifolate drug aminopterin results in greatly enhanced synthesis of Hcy-thiolactone by L. luteus plants. Methionine inhibits the synthesis of Hcy-thiolactone in L. luteus, suggesting involvement of methionyl-tRNA synthetase. Consistent with this suggestion is our finding that the plant Oryza sativa methionyl-tRNA synthetase, expressed in Escherichia coli, catalyzes conversion of Hcy to Hcy-thiolactone. We also show that Hcy is a component of L. luteus proteins, most likely due to facile reaction of Hcy-thiolactone with protein amino groups. In addition, L. luteus possesses constitutively expressed, highly specific Hcy-thiolactone-hydrolyzing enzyme. Thus, Hcy-thiolactone and Hcy bound to protein by an amide (or peptide) linkage (Hcy-N-protein) are significant components of plant Hcy metabolism. (+info)
Self-aggregation of legume seed storage proteins inside the protein storage vacuoles is electrostatic in nature, rather than lectin-mediated.
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Conglutins are multisubunit, glycosylated, major storage proteins present in Lupinus seeds that self-aggregate in a calcium/magnesium-dependent manner. Two of these globulins exhibit lectin activity. The 210 kDa globulin derived from beta-conglutin that accumulates in Lupinus cotyledons during germination was used as a model protein to establish whether the self-aggregation process is electrostatic in nature or lectin-mediated. This protein binds in a very strong manner to chitin and recognizes a variety of glycoproteins including immunoglobulins G. Several compounds were tested for their inhibitory effect on the cation-dependent self-aggregation process. Sialic acid and phytin were the most effective whereas chitin and mucin were totally ineffective. The inability of the oligosaccharidic side chains of the 210 kDa protein, beta-conglutin and immunoglobulin G to interfere with the aggregation strongly supports the view that Ca/Mg are electrostatically involved in the in vitro self-aggregation of Lupinus globulins. The results suggest that calcium and magnesium ions are also electrostatically involved in vivo in the macromolecular aggregation of legume seed storage proteins, ensuring their efficient packing inside the protein storage vacuoles. This mechanism is responsible for the typical insolubility of legume globulins in water. (+info)
Nylon filter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus deficiency.
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White lupin (Lupinus albus) adapts to phosphorus deficiency (-P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. In an effort to better understand the molecular events mediating these adaptive responses, we have isolated and sequenced 2,102 expressed sequence tags (ESTs) from cDNA libraries prepared with RNA isolated at different stages of proteoid root development. Determination of overlapping regions revealed 322 contigs (redundant copy transcripts) and 1,126 singletons (single-copy transcripts) that compile to a total of 1,448 unique genes (unigenes). Nylon filter arrays with these 2,102 ESTs from proteoid roots were performed to evaluate global aspects of gene expression in response to -P stress. ESTs differentially expressed in P-deficient proteoid roots compared with +P and -P normal roots include genes involved in carbon metabolism, secondary metabolism, P scavenging and remobilization, plant hormone metabolism, and signal transduction. (+info)
Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases.
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Dinucleoside 5',5"'- P (1), P ( n )-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5'- O -phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5'- O -phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap(4)A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5'- O -phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5'- O -phosphorylated counterparts. 1,4-Di(adenosine-5'- O -phosphorothio) erythritol appeared to be the strongest inhibitor of ( asymmetrical ) Ap(4)A hydrolases (EC 3.6.1.17) from both lupin and human, with K (i) values of 0.15 microM and 1.5 microM respectively. Of eight adenosine-5'- O -phosphorylated polyols, 1,4-di(adenosine-5'- O -phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5'- O -phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5'- O -phosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic ( symmetrical ) Ap(4)A hydrolase (EC 3.6.1.41) so far reported. The estimated K (i) values were 0.04 microM and 0.08 microM respectively. All of these inhibitors were competitive with respect to Ap(4)A. These new selectively acting Ap(4)A analogues should prove to be valuable tools for further studies of Ap(4)A function and of the enzymes involved in its metabolism. (+info)