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Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves.

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.

Abscisic Acid↗

Ion-specific mechanisms of osmoregulation in bean mesophyll cells.

Transient kinetics of net H+, K+, Ca2+, and Cl- fluxes were measured non-invasively, using an ion-selective microelectrode technique, for bean (Vicia faba L.) leaf mesophyll in response to 150 mM mannitol treatment. In a parallel set of experiments, changes in the plasma membrane potential and the total proline content in leaves were monitored. Regardless of the ionic composition of the bath solution, hyperosmotic stress caused a significant increase in the K+ and Cl- uptake into mesophyll cells. At the same time, no significant proline changes were observed for at least 16 h after the onset of stress. Experiments with inhibitors suggested that potassium inward rectifier (KIR) channels, exhibiting mechanosensitive properties and acting as primary receptors of osmotic stress, are likely to be involved. Due to the coupling by membrane potential, changes in K+ and Cl- transport may modify activity of the plasma membrane H+-pump. Such coupling may also be responsible for the mannitol-induced oscillations (period of about 4 min) in net ion fluxes observed in 90% of plants. Calculations show that influx of K+ and Cl- observed in response to hyperosmotic treatment may provide an adequate osmotic adjustment in bean mesophyll, which suggests that the activity of the plasma membrane transporters for these ions should be targeted to improve osmotolerance, at least in this crop.

Calcium↗

Removal of O6-methylguanine from plant DNA in vivo is accelerated under conditions of clastogenic adaptation.

Previously it was shown that the clastogenic efficiency of high doses of alkylating agents in plant root meristems can be reduced significantly by conditioning pretreatment with either a low dose of the same agents, a sublethal heat shock, or heavy metal salts. The molecular mechanisms responsible for these protective effects are still unclear. Here we report on the quantification of O6-methylguanine [O6-MeG] by immuno-slot-blot analysis in DNA of root tip meristems of field bean (Vicia faba) seedlings under conditions of clastogenic adaptation. When root tips were pretreated with a low, conditioning dose of N-methyl-N-nitrosourea (MNU, 10(-4) M, 1 hour) 2 hours before exposure to a high dose of the same clastogen (10(-3) M, 1 hour), the frequency of chromatid aberrations was reduced by more than 50% at a recovery time of 1 B hours, as compared to treatment with the high dose alone. The same was observed when conditioning pretreatment was by a sublethal heat shock [10 minutes, 40 degrees C] or a heavy metal salt (Cd(NO)3, 10(-7) M, 1 hour). The frequency of O6-MeG immediately after exposure to a conditioning and a subsequent challenge treatment was reduced by 43% as compared to treatment with only the high dose. At a recovery time of 18 hours the corresponding frequency of adducts was reduced by 68.3% (related to the initial level) after treatment with the high dose alone, and by 81.3% under adaptive conditions. Sublethal heat shock or heavy metal salt used as conditioning pretreatments also resulted in a decrease of adducts immediately after treatment with the challenge dose. From these data and from prevention of the effects by pretreatment with cycloheximide or O6-benzylguanine we conclude that under conditions of clastogenic adaptation O6-MeG is more efficiently removed from the DNA, presumably by induction of an alkyl acceptor protein such as O6-methylguanine-DNA methyltransferase [MGMT]. This could explain the observed protective effects (clastogenic adaptation.

Adaptation, Physiological↗

Amino acid composition and biological effects of supplementing broad bean and corn proteins with Nigella sativa (black cumin) cake protein.

The biological effects of supplementing broad bean (Vicia faba) or corn (Zea maize) meal protein with black cumin (Nigella sativa) cake protein as well as their amino acid composition were investigated. The percentage of total protein content of Nigella cake was 22.7%. Lysine is existent in abundant amounts in faba meal protein, while leucine is the most abundant in corn meal protein (chemical score = 156) and valine is higher in Nagella cake protein. compared with rats fed sole corn or faba meal protein, substitution of 25% of corn or faba meal protein with Nigella cake protein in the diet remarkably raised the growth rate of rats and resulted in significant higher levels of rat total serum lipids and triglycerides. Also, the supplemented diet caused significant increases in serum total protein and its two fractions albumin and globulin and insignificantly increase the activity of serum phosphatases and transaminases within normal ranges. The supplementation did not have any adverse nutritional effects in the levels of lipid fractions in the serum.

Alanine Transaminase↗

A comparison of acute mortality and population growth rate as endpoints of toxicological effect.

The objective of this study was to determine how closely acute (72-hr) lethal concentration estimates developed from probit analysis compared to the demographic toxicological endpoints, net reproductive rate (Ro), the intrinsic rate of increase (rm), and realized fecundity (Ux), in terms of predicting effects of pesticides on populations. Lethal and sublethal effects of the insecticide imidacloprid on the arthropod Acyrthosiphon pisum Harris (pea aphid) were determined for populations exposed to foliar-sprayed broad bean Vicia faba L. (variety Banner). An examination of Ro indicated that sublethal effects were occurring that reduced reproduction. However, by looking at the mean number of offspring produced per surviving female and Ux, it was determined that the reduction in Ro was entirely due to acute mortality and a reduction in life span. Also, exposure to increasing concentrations of imidacloprid did not cause a shift in either the day of initial reproduction or the day of peak reproduction. Therefore, this pesticide caused no sublethal effects on reproduction and, as such, a lethal concentration estimate should have been a good predictor of effect at the population level. However, the 72-hr lethal concentration estimate was not a good predictor of effect of this pesticide on population growth. Populations exposed to the 72-hr LC60 were able to maintain rates of population increase (rm = 0.224) similar to those of the control (rm = 0.295). The data indicate that the reason for the discrepancy between acute lethal concentration estimates and population growth was that surviving individuals were able to sustain heightened rates of reproduction following acute exposure to imidacloprid. The ability of surviving individuals to maintain these high reproductive rates allowed them to compensate for losses and act as reservoirs for future reproduction. It is not possible, using acute mortality estimates alone, to predict this "reservoir effect," and therefore not possible to predict how a population's growth rate will respond or change based on this endpoint. Thus this would suggest that the assessment of a xenobiotic based solely on acute mortality estimates will lead to flawed conclusions about a population's exposure response.

Animals↗

Localization of seed protein genes on flow-sorted field bean chromosomes.

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using 'nested' primers. The genes are located within defined regions of chromosome I (USP = unknown seed protein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes psi 1) and V (legumin A genes and pseudogenes psi 1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.

Base Sequence↗

The nutritive value of some selected Tanzanian plant food sources.

The chemical composition of different varieties of field beans (Vicia faba L.), cowpeas (Vigna unguiculata), groundnuts (Voandzeia subterranea), and sorghum (Sorghum vulgare) were determined. Nutrients analysed in these materials included amino acids, starch, sugar, fibre, minerals and antinutritional factors. All the materials were further tested in balance trials with rats; true protein digestibility (TD), biological value (BV), net protein utilization (NPU) and digestible energy (DE) were evaluated. Based on the chemical analyses as well as on the biological data, the nutritive value of plant materials between species as well as between varieties within species differed considerably. Therefore, more quality evaluation studies along with plant breeding programmes are needed to select the most promising varieties from the nutritional point of view.

Amino Acids↗

Formulation, acceptability, chemical composition and in vitro digestibility of novel snack food and meat ball analogue.

Potato purée and wheat flour were blended at a ratio of 7:1 w/w with 2 parts per weight of soybean protein concentrate (SPC) and/or faba bean-protein micellar mass (PMM). Two snack foods were formulated and deep fried. Spices, onion, parsley and chicken stock were added to the aforementioned two recipes to formulate meat ball analogues. Data revealed good acceptability of all products. Moreover, they can be considered as a good source for some essential amino acids; protein (approximately 26%); lipids (approximately 20%); carbohydrates (approximately 48%) and minerals. The in vitro digestibility ranged between 61.0% and 66.9%.

Amino Acids↗

[Examination and composition of some legume seeds (author's transl)].

The chemical composition of some legume seeds is reviewed. It was found that soy (Glycine soja), field beans (Vicia faba), Vicia sativa and Lens culingris (red seeds) have the highest protein content. There were only minor differences in amino acid composition except for glutamic acid. The lysine content of legume seeds was high. Lysine is the most limiting essential amino acid in cereal proteins, therefore cereal products can be improved nutritionally by addition of legume seed flour. Examination of polar lipids by thin layer chromatography revealed that lecithin, phosphatidyl ethanolamine and phosphatidyl inositol were the major components. However, Lupinus angustifolius contained two unknown polar lipid classes.--In contrary to previous literature the ripe legume seeds contained only a very small amount of galactosyl diglycerides; however legume seeds contain higher amounts of the compounds. The conection between biosynthesis of linolenic acid and the presence of galactosyl diglycerides in higher plants is discussed.

Amino Acids↗

Histone H4 acetylation in plant heterochromatin is altered during the cell cycle.

Using polyclonal antibodies directed against acetylated isoforms of histone H4 (H4 acetylated at lysine positions 5, 8, 12, 16 and H4 tetraacetylated), indirect immunofluorescence revealed hyperacetylation for all H4 variants at the nucleolus organizer region (NOR) of metaphase chromosomes of the field bean Vicia faba. The transcriptionally inactive and late-replicating heterochromatin regions proved to be hypoacetylated at lysine positions 5, 8 and 12. The remaining chromatin showed average fluorescence. These patterns were altered when deacetylase was blocked by exposure of root tip meristems to trichostatin A for more than 2 h prior to fixation. Under these conditions, all lysine positions, except lysine 8, appeared to be hyperacetylated at the NOR and in addition at the prominent heterochromatin domains. This observation represents a hitherto unique switch of histone acetylation pattern during the cell cycle. This is apparently caused by deposition of acetylated H4. Ac5, 12 and 16 or by acetylation directly after replication, which later on becomes reduced (H4.Ac16) or even reversed (H4.Ac5 and 12) by deacetylase before cells enter mitosis.

Acetylation↗

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.

Carrier Proteins↗

Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization.

Although the presence of a matrix in plant nuclei has been reported, major questions remain about its structural and biochemical features. We have used an intermediate filament antibody of broad specificity to explore whether Daucus carota (carrot) nuclei and nuclear matrices contain intermediate filament/lamin antigens and, if so, where specifically they are localized. SDS-PAGE and Western blotting revealed two bands, at 60 and 65 kDa, that were highly immunoreactive with the intermediate filament antibody (IFA) of Pruss et al. (1981, Cell 27, 419-428). This pattern was observed consistently, not only with carrot cell-free nuclei and nuclear matrices, but also with nuclear preparations from Vicia faba (broad bean) and Pisum sativum (pea). Immunofluorescence studies with whole carrot nuclei localized the IFA antigens to the nucleoplasm and disclosed no accentuated peripheral labeling. Agarose-embedded nuclear matrices showed not only fluorescence throughout the nucleoplasm but also heavy labeling surrounding the nucleoli and suggestions of peripheral labeling. At the ultrastructural level, immunogold results from pre- and postembedment treatments supported the conclusion that IFA antigens occur throughout the nucleoplasm, with possibly a slight concentration at the periphery. These combined results provide substantial evidence that plant nuclei and their matrices possess at least two major intermediate filament antigens with molecular weights characteristic of animal lamins. Whether or not these antigens represent plant lamins, their nonperipheral localization hints at significant differences among the eukaryotic kingdoms in nuclear organization.

Antigens↗