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[Comparative studies on the reactive sites against trypsin of some inhibitors from phaseolus coccineus and phaseolus vulgaris (author's transl)].

Three of the trypsin chymotrypsin inhibitors from the seeds of runner beans (Phaseolus coccineus L.), PCI 3,4(2), and 5, and three of the inhibitors from the seeds of french beans (Phaseolus vulgaris var. nanus), PVI 3, 4, and 5, contain a lysine residue in the reactive site against trypsin. One of the inhibitors from Phaseolus coccineus, PCI 2, contains an arginine residue there. All seven Phaseolus inhibitors investigated are double headed.

Lysine↗

The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants.

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.

Arabidopsis↗

Tannins, trypsin inhibitors and lectin cytotoxicity in tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans.

This study compared the levels of antinutritional components and cytotoxic effect of extracts, from tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Antinutritional factors were evaluated by determining their effect on the viability of epithelial cells isolated from rat small intestine. The protein and carbohydrates content were similar in all the genotypes studied (20 and 60%, respectively). Common beans presented higher content of trypsin inhibitors, tannins and lectins than tepary beans. There was not a significant correlation between tannins and cooking time. However, water absorption and cooking time correlated significantly (p < 0.05). Considerable variation was observed in lectin activity (1302-18161 Ul/mg) of extracts from different beans. Tannins, lectins, trypsin inhibitors and fat content differed between bean varieties whereas protein content was similar. The percent cellularity on rat epithelial cells was significantly different among protein extracts from different bean cultivars and ranged between 53.5% and 87.4% (p < 0.05). These results suggest that the incorporation of tepary beans in the diet would not alter the current nutritional contribution of common beans or introduce adverse toxic effects. The agronomic characteristics of tepary beans make them attractive for cultivation. However, the harder to cook phenomenon may be a limiting factor that needs further consideration.

Animals↗

The diversity of Phaseolus-nodulating rhizobial populations is altered by liming of acid soils planted with Phaseolus vulgaris L. in Brazil.

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (Phaseolus vulgaris L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with Rhizobium tropici IIB and Rhizobium leguminosarum bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on "species" types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of R. tropici IIB pattern types contained the largest number of ITS groups. In contrast, there were more R. leguminosarum bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS ("strain") groups within R. leguminosarum bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with R. tropici IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of Phaseolus-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.

Brazil↗

Molecular research on the genetic diversity of Polish varieties and landraces of Phaseolus coccineus L. and Phaseolus vulgaris L. using the RAPD and AFLP methods.

The aim of our research was to evaluate the genetic diversity among 25 commercial varieties registered in Poland and 14 landraces of Phaseolus vulgaris var. nanus Asch. (the dwarf common bean) and Phaseolus coccineus L. (the runner bean) maintained in the National Centre of Plant Genetic Resources in Radzików. An additional goal of this study was to compare the precision and efficiency of two techniques of PCR (RAPD and AFLP), used to estimation the genetic diversity of bean. The breeding varieties of bean were registered in the period between 1950 and 2000. The landraces, collected during expeditions conducted from 1985 to 1988, mainly originated from the eastern and southern part of Poland. In the plant genetic diversity research of RAPD and AFLP markers are commonly used. Complex electrophoresis pictures of DNA fragments were taken, and revealed a considerable polymorphism. The polymorphic fragments were obtained on the basis of 6 differentiating primers using the RAPD method and 15 differentiating primers using the AFLP method. P. vulgaris and P. coccineus accessions formed distinct groups. Each of the RAPD and AFLP analyses allowed for the unique distinguishing of all accessions.

Base Sequence↗

Immunosuppressive activity of Phaseolus coccineus and Phaseolus vulgaris extracts in mice.

Lectin mixtures from 10 samples of Phaseolus coccineus and Phaseolus vulgaris beans were used as immunosuppressive agents in the sheep red blood cell (SRBC) antibody response of the mouse. Some bean samples contain lectins capable of inducing several times better suppression of humoral immunity than concanavalin A and phytohemagglutinin tested before in the same immune system. High dilutions of the 10 bean extracts were shown to agglutinate mouse, rabbit and sheep erythrocytes in vitro but the titers and "specificities" did not correlate with the immunosuppressive potency and selectivity displayed by the same extracts in mice. Two immunosuppressive bean extracts, assayed in lymphocyte cultures, were found to stimulate DNA synthesis of murine spleen T cells. This interaction appears to be a necessary step for the reduction of antibody synthesis to SRBC in the whole animal.

Animals↗

The cytoplasmic male-sterility (CMS) determinant of common bean is widespread in Phaseolus coccineus L. and Phaseolus vulgaris L.

To identify regions of the mitochondrial genome that potentially could specify cytoplasmic male sterility (CMS) in Phaseolus coccineus (including P. polyanthus), and to define differences amongst P. coccineus lines, mitochondrial (mt)DNA restriction patterns and Southern blots of total DNA from sterile and fertile lines were analysed. By restriction endonuclease mapping we isolated a region which was specific to CMS lines flanking an F1-ATPase alpha-subunit (atpA) gene. DNA sequence analysis of this region showed 99.9% homology to the region previously isolated from P. vulgaris CMS Sprite. A high frequency of plants carrying the CMS-fragment was observed in a wild Phaseolus population, perhaps explaining the occurrence of inter- and intra-specific gene flow observed in the autogamous species P. vulgaris.

Blotting, Southern↗

Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules.

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS(n1)) and a band (GS(n2)) similar to the single band (GS(r)) present in root extracts. In nodule extracts of P. lunatus, the GS(n1) band was detected, but the GS(n2) band was barely detectable. In contrast to P. vulgaris, the GS(n2) band and the GS(r) band of P. lunatus appeared to be different. The electrophoretic mobility of the GS(n1) band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS(n1) activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS(n1) of increasing electrophoretic mobility during the course of nodule development.

Journal Article↗

Genetic Structure of Rhizobium etli biovar phaseoli Associated with Wild and Cultivated Bean Plants (Phaseolus vulgaris and Phaseolus coccineus) in Morelos, Mexico.

The genetic structure of Rhizobium etli biovar phaseoli was determined for five populations in three different locations in the state of Morelos, Mexico, by using starch gel electrophoresis for five to nine polymorphic loci. Two populations were sampled during two different years from nodules of cultivated and wild common bean plants (Phaseolus vulgaris). The three other populations were associated with wild runner beans (P. coccineus) and sampled during 1988. The Rhizobium populations differ genetically both among sites and among populations within the same site in different years, as shown by differences in allelic frequencies, genetic differentiation analysis, and differences in electrotypes. The total genetic diversity for the five populations during 1988 was H = 0.487; there were also high levels of genetic variation within each population. We found the highest linkage disequilibrium in a global analysis for all the populations. At a local scale, we also found significant linkage disequilibrium in two populations, although the distribution of the D' suggest some recombination at a local scale. The other three rhizobium populations exhibit low linkage disequilibrium. A cluster analysis (UPGMA) of pairwise genetic distances showed that bacteria isolated from most wild Phaseolus spp. are grouped by population, whereas those obtained from cultivated P. vulgaris are very heterogeneous. The analysis of the genetic structure of Rhizobium strains may allow the identification of strains that are naturally well adapted to a wide range of different environments, which may be useful for agricultural purposes or as a starting point for developing improved Rhizobium strains.

Journal Article↗

[Comparative study of the chemical composition and nutritive value of runner bean (Phaseolus coccineus) and of common bean (Phaseolus vulgaris)].

Scarlet beans (Phaseolus coccineus) is an important food grain legume in the diet of rural populations living in the highlands of México, Guatemala and other countries of Latin America. The present study was conducted to obtain more chemical and nutritive data on this grain legume, because of its importance in agricultural production systems and due to the role it plays in rural diets. Ten samples were purchased in the highland markets of Guatemala, to compare their physical, chemical and nutritive characteristics with three samples of common beans (Phaseolus vulgaris). A sample of 10 kg was used for nutritional studies, fed alone and in mixtures with maize. The results indicated that P. coccineus has greater weight as compared to P. vulgaris (0.74 vs. 0.18 g) and are larger in size (0.60 vs. 0.14 cc/grain). Seed coat percentage was 10.2% for P. coccineus as compared to 9.0% for P. vulgaris. The cooking time was 231 minutes for P. coccineus and 180 minutes for common beans. Seed coat thickness and permeability are different when comparing P. coccineus with P. vulgaris, since at 24 hr soaking time, P. coccineus absorbed 80% of its water weight as compared to 100% for common beans. In chemical composition) small differences were found between P. coccineus and P. vulgaris. The first had slightly more ether extract, crude fiber and ash content than the second. Amino acid content among the six samples of P. coccineus was quite variable, but its pattern was rich in lysine and limiting in sulfur amino acids. Cooking time at 3 hr gave a higher NPR than when cooked at atmospheric pressure for 5 hr, with protein values relative to casein of 69.2 and 60.0%, respectively. P. coccineus protein quality was improved by methionine supplementation, and it appeared to be better digested than that of P. vulgaris. Mixtures of high protein quality were obtained with 15% P. coccineus and 85% maize while common beans and maize mixed in a 30 to 20 ratio showed maximum protein efficiency. The digestibility of the P. coccineus/maize mixtures was higher than that of common beans and maize.

Amino Acids↗

Comparative study of enzymes related to proline metabolism in tepary bean (Phaseolus acutifolius) and common bean (Phaseolus vulgaris) under drought and irrigated conditions, and various urea concentrations.

There are several mechanisms used by plants for survival in adverse environments such as drought, high temperature and salinity. The objective of this study was to evaluate the drought tolerance of tepary bean as a function of biochemical processes linked to isozyme synthesis and changes in enzymatic activity related to proline metabolism. Mature seeds of common beans var. flor de mayo, Phoseolus vulgaris and tepary beans Phaseolus acutifolius were grown under two water conditions (irrigation and drought), and four levels of urea. Vertical electrophoresis and spectrophotometric techniques were used to evaluate protein patterns, glutamate dehydrogenase (GDH), proline oxidase (PO) and pyrroline-5-carboxylate reductase (P5C reductase) enzyme activities. These enzymes were studied because they are directly related to protein synthesis. Electrophoretic patterns showed more proteins in tepary beans than in common beans with limited irrigation. GDH showed only one isozyme, with a molecular weight between 240) to 270 kDa. A decrease in PO activity was observed in common beans under drought stress with a value of 237 micromol/min, in comparison to irrigation conditions of 580 micromol/min. GDH and P5C reductase enzymes have had higher activity in common beans than in tepary beans under water stress. There was a significant difference only in glutamate dehydrogenase enzyme with respect to urea level. The results suggest that drought tolerance of tepary beans is due to biochemical processes related to proline metabolic enzymes.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

Cytokinin-Active Ribonucleosides in Phaseolus RNA: I. IDENTIFICATION IN tRNA FROM ETIOLATED PHASEOLUS VULGARIS L. SEEDLINGS.

The cytokinin-active ribonucleosides present in tRNA from etiolated Phaseolus vulgaris L. seedlings have been isolated and identified as cis-ribosylzeatin, 2-methylthio-ribosylzeatin, and N(6)-(Delta(2)-isopentenyl)-adenosine. The structures of the compounds were established on the basis of their chromatographic properties and the mass spectra of their permethylated and perdeuteromethylated derivatives. Cis-ribosylzeatin was the major cytokinin-active constituent of tRNA from this source.

Journal Article↗

Comparative Water Relations of Phaseolus vulgaris L. and Phaseolus acutifolius Gray.

Leaf area expansion, dry weight, and water relations of Phaseolus vulgaris L. and P. acutifolius Gray were compared during a drying cycle in the greenhouse to understand the characteristics which contribute to the superior drought tolerance of P. acutifolius. Stomates of P. acutifolius closed at a much higher water potential than those of P. vulgaris, delaying dehydration of leaf tissue. P. acutifolius had a more deeply penetrating root system, which also contributes to its drought tolerance. Root-shoot ratios did not differ between the two species either under well watered or water stressed conditions. Leaf osmotic potential was also similar in the two species, with no apparent osmotic adjustment during water stress. These results indicate that P. acutifolius postpones dehydration and suggest that sensitive stomates and a deeply penetrating root system are characteristics which, if incorporated into cultivated beans, might increase their drought tolerance.

Journal Article↗

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L.

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25 degrees C and 32 degrees C. Mitochondria isolated from P. vulgaris plants grown at 32 degrees C had reduced electron transport and were substantially uncoupled. Growth at 32 degrees C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25 degrees C grown plants. Mitochondria isolated from 25 degrees C grown P. vulgaris plants measured at 42 degrees C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42 degrees C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.

Journal Article↗

Phaseolin nucleotide sequence diversity in Phaseolus. I. Intraspecific diversity in Phaseolus vulgaris.

Most information about the molecular biology of phaseolin, the major seed storage protein in Phaseolus vulgaris, has been obtained from the T-type phaseolin, which is characteristic of the Andean gene pool of the species. In the work reported here, two cDNA clones for the S-type phaseolin representing the other major, Middle American gene pool were isolated and sequenced. Analysis of the DNA sequences revealed the presence of two subtypes of S phaseolin, alpha and beta, depending on the presence or absence, respectively, of a 27-bp direct repeat. These are similar to the alpha- and beta-phaseolin subtypes found in the Andean, T phaseolin; however, the additional 15-bp direct repeat also found in the T alpha-phaseolin gene type was apparently absent from the S alpha-phaseolin genes. The overall sequence identity was greater between the alpha or beta subtypes of different gene pools than between the alpha or beta subtypes within gene pools. This implies that the gene subtypes were formed prior to the formation of the two major gene pools of P. vulgaris. Analysis of the putative amino acid sequence revealed that both the 'Sanilac' phaseolin subtypes contained an additional methionine, however, not at the same site. This opens the possibility of increasing the nutritionally limiting methionine level in phaseolin either through protein engineering or by screening accessions for recombinant phaseolin sequences that combine both substitutions.

Amino Acid Sequence↗

Simultaneous anterograde labelling of two afferent pathways to the same target area with Phaseolus vulgaris leucoagglutinin and Phaseolus vulgaris leucoagglutinin conjugated to biotin or dinitrophenol.

Anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and PHA-L conjugated to either biotin or 2,4-dinitrophenol were used to simultaneously label two populations of axons converging onto the same target area. Using the rat hippocampus as a test system, the septohippocampal and contralateral hilar afferents were labelled with the tracers. Double immunohistochemical procedures and either nickel-enhanced 3,3'-diaminobenzidine (DAB) reaction (blue-black colour) or DAB alone (brown colour) were used to produce contrasting colours for the different tracers in the same section. Both the biotinylated PHA-L and PHA-L conjugated with dinitrophenol were used successfully as anterogradely transported axonal tracers. They produced extensive axonal labelling in the hippocampal formation. Excellent double labelling could be produced with the simultaneous application of PHA-L and biotinylated PHA-L. Biotinylated PHA-L was visualized by a sequence of avidin-biotinylated peroxidase complex (ABC), biotinylated goat antiavidin and ABC again using nickel-enhanced DAB as chromogen yielding a blue-black reaction endproduct. PHA-L alone was detected by the unlabelled antibody enzyme (PAP) method using goat antibodies to PHA-L, and DAB as chromogen for the peroxidase reaction, resulting in brown axons. The combination of biotinylated PHA-L and DNP-conjugated PHA-L gave similar results, although the sensitivity of detection by the latter procedure was inferior to that obtained with the other tracers. These protocols permitted visualization of axons of different origin, together with their terminals, either in a blue-black or brown colour, and also allowed the demonstration of overlapping inputs in strata radiatum and lacunosum moleculare of the hippocampus and stratum moleculare and hilus of the dentate gyrus.

Animals↗

Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean, Phaseolus vulgaris.

8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins.

Adenine↗

In vitro digestibility of proteins in black gram (Phaseolus mungo) and green gram (Phaseolus radiatus) papads.

'Papads', a traditional food of India, were prepared with decorticated black gram and green gram flours and toasted, microwaved or deep fat fried. Toasting was done by holding the papads over direct flame for 1-2 min. Sharp carousal microwave oven was used for microwaving at high temperature for 30-40 s. Deep fat frying was done by immersing the product in preheated refined groundnut oil for 5-10 s. In vitro digestibility of proteins in prepared papads was investigated by pepsin and pepsin + pancreatin. The percent protein hydrolysed in black gram papads was 80.5, 75.3 and 72.5 for toasted, microwaved and fried papads respectively. They were not significantly different from each other. Green gram papads exhibited significantly higher hydrolysis in microwaved product (79.3%) when compared to toasted (65.2%) or fried products (65.0%). Pepsin digestibilities were not influenced to a significant extent by cooking method and were in the range of 51.4-54.6% for black gram papads and 34.4-35.6% for green gram papads.

Cooking↗