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Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library.

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.

Journal Article↗

Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad.

Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-amylase inhibitor (alpha AI). Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein alpha AI, which exerts its biological activity by making a 1:1 complex with alpha-amylase. alpha-amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn77 to form an active molecule. Comparison of the amino acid sequence of the bean alpha AI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp188 might be involved in the inhibitory site. When a three-dimensional model of the bean alpha AI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn77. To test this site hypothesis, mutants of alpha AI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco. The mutant proteins R74N and WSY188-190GNV, as well as the double mutant, were inactive as inhibitors. These findings suggest that the active site of alpha AI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77 may be necessary to bring these residues in close proximity.

Amino Acid Sequence↗

Activation of bean (Phaseolus vulgaris) alpha-amylase inhibitor requires proteolytic processing of the proprotein.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the alpha-amylases of mammals and insects. This alpha-amylase inhibitor (alpha AI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M(r)) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to alpha AI recognize large (M(r) 30,000-35,000) polypeptides as well as typical alpha AI processing products (M(r) 15,000-18,000). Alpha AI activity was found in all extracts that had the typical alpha AI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant alpha AI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-alpha AI when the gene is expressed in tobacco. When pro-alpha AI was separated from mature alpha AI by gel filtration, pro-alpha AI was found not to have alpha-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.

Adsorption↗

[Hardening and softening phenomena in beans: technological alternatives].

The effect of accelerated hardening and soaking solutions on cooking time and microstructure of common bean (Phaseolus vulgaris) was studied. Two varieties (Canario and Mayocoba) were grown in the same location. Three hardening procedures were used: 1) End A. Soaking in acetate buffer, pH = 4.0 at 37 degrees C for 5 hs, 2) End B. Storage at 37 degrees C, 100% RH for 28 days and, 3) End C storage at 13-33 degrees C, 76% RH for 120 days. The salt solutions used for soaking were: Soln 1 (1% NaCl+0.75% NaHCO3) and Soln 2 (0.75% NaHCO3). Cooking times were determined using a Mattson bean cooker. In both varieties, the three hardening procedures decreased (38-50%) cotyledons water holding capacity and increased significantly (2-4 times) cooking times. During soaking in salt solutions hardened beans reached maximum water absorption in four hours. Soaking in salt solutions decreased drastically (2.6-10.6 times) cooking times. Fresh, hardened and softened seeds were examined by light microscopy, observing ultrastructural differences among them. The methods used in this research might well represent the central components of an industrial technological procedure for the utilization of hardened beans.

Absorption↗

Bean dwarf mosaic virus BV1 protein is a determinant of the hypersensitive response and avirulence in Phaseolus vulgaris.

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differeBean dwarf mosaic viru certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.

Carrier Proteins↗

Shotgun identification of proteins from uredospores of the bean rust Uromyces appendiculatus.

We are interested in learning more about the proteome of Uromyces appendiculatus, the fungus that causes common bean rust. Knowledge of the proteins that differentiate life-cycle stages and distinguish infectious bodies such as uredospores, germlings, appressoria, and haustoria may be used to define host-pathogen interactions or serve as targets for chemical inhibition of the fungus. We have used 2-D nanoflowLC-MS/MS to identify more than 400 proteins from asexual uredospores. A majority of the proteins appear to have roles in protein folding or protein catabolism. We present a model by which an abundance of heat shock proteins and translation elongation factors may enhance a spore's ability to survive environmental stresses and rapidly initiate protein production upon germination.

Amino Acid Sequence↗

Molecular cloning of the cDNA encoding aspartate aminotransferase from bean root nodules and determination of its role in nodule nitrogen metabolism.

A cDNA clone encoding aspartate aminotransferase (PVAAT-2) (EC 2.6.1.1) was isolated from the common bean Phaseolus vulgaris nodule cDNA library. The nucleotide sequence analysis of the full-length cDNA allowed its identification by comparison with sequence databases. The amino acid sequence of the bean PvAAT-2 showed high similarity with the AAT-2 isoforms described in other leguminous plants. The amino-terminal region of the PvAAT-2 contains a sequence, which shares common features of plastid transit peptides. Southern blot analysis showed that the PvAAT-2 clone is encoded by a single gene in the P. vulgaris genome. Analysis of the PvAAT-2 mRNA levels suggests that the expression of this gene is nodule enhanced. The PvAAT-2 transcript is more abundant in nodules with increased synthesis of amides and is down-regulated in conditions where ureides accumulate. When plants were supplemented with ureides or with amides, PvAAT-2 expression was reduced, while it was not affected when plants were treated with allopurinol, an inhibitor of ureide synthesis. On the other hand, the expression of asparagine synthetase (another enzyme involved in the synthesis of amides) is not affected either by ureides or amides. These data suggest a role for AAT-2 in the mechanism involved in the synthesis of nitrogen compounds in bean nodules.

Acetylene↗

Bipartite geminivirus host adaptation determined cooperatively by coding and noncoding sequences of the genome.

Bipartite geminiviruses are small, plant-infecting viruses with genomes composed of circular, single-stranded DNA molecules, designated A and B. Although they are closely related genetically, individual bipartite geminiviruses frequently exhibit host-specific adaptation. Two such viruses are bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV), which are well adapted to common bean (Phaseolus vulgaris) and Nicotiana benthamiana, respectively. In previous studies, partial host adaptation was conferred on BGMV-based or TGMV-based hybrid viruses by separately exchanging open reading frames (ORFs) on DNA A or DNA B. Here we analyzed hybrid viruses in which all of the ORFs on both DNAs were exchanged except for AL1, which encodes a protein with strictly virus-specific activity. These hybrid viruses exhibited partial transfer of host-adapted phenotypes. In contrast, exchange of noncoding regions (NCRs) upstream from the AR1 and BR1 ORFs did not confer any host-specific gain of function on hybrid viruses. However, when the exchangeable ORFs and NCRs from TGMV were combined in a single BGMV-based hybrid virus, complete transfer of TGMV-like adaptation to N. benthamiana was achieved. Interestingly, the reciprocal TGMV-based hybrid virus displayed only partial gain of function in bean. This may be, in part, the result of defective virus-specific interactions between TGMV and BGMV sequences present in the hybrid, although a potential role in adaptation to bean for additional regions of the BGMV genome cannot be ruled out.

Adaptation, Physiological↗

Status of free radicals in Indian monsooned coffee beans gamma-irradiated for disinfestation.

Free radicals in two cultivars of Indian monsooned coffee beans, gamma-irradiated for hygienic and quarantine purposes, were examined by entrapping the small amount of samples in potassium chloride powder in ESR quartz tubes. In contrast to a prominent free radical signal at g = 2.002, observed in spermoderm (silver skin) and cotyledon (whole seed without skin) parts of normal coffee beans, the same was not discernible in monsooned coffee bean parts of both cultivars. The ESR signal was found to be more prominent in the spermoderm than in the whole seed portion of the normal coffee beans. Common practices of roasting and powdering were found to generate quantitatively more free radicals in coffee beans than gamma-irradiation alone. Phenols, contributing maximally to observed free radical signals in coffee beans, were significantly different in normal and monsooned coffee beans. These observations on insignificant free radical population in irradiated monsooned coffee beans may be attributed to their inherent possession of high water activity, favoring decay of free radicals produced. Textural studies with monsooned coffee beans, before and after mild heat treatments, supported these findings.

Chlorogenic Acid↗

Generalized Induction of Defense Responses in Bean Is Not Correlated with the Induction of the Hypersensitive Reaction.

Transcripts for phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, and chitinase accumulated in common bean after infiltration with the Pseudomonas syringae pv tabaci Hrp- mutant Pt11528::Hrp1, even though a hypersensitive reaction did not occur. The temporal pattern of this transcript accumulation was similar to that seen after infiltration with wild-type P. s. tabaci Pt11528, which resulted in a hypersensitive reaction. Escherichia coli DH5[alpha], P. fluorescens Pf101, heat-killed Pt11528 cells, and Pt11528 cells treated with protein synthesis inhibitors also induced accumulation of defense transcripts but not a hypersensitive reaction. In contrast, these transcripts were not detected in plants infiltrated with water or P.s. pv phaseolicola NPS3121, a compatible pathogen that causes halo blight. Phytoalexins were produced in bean after infiltration with Pt11528, Pt11528::Hrp1, Pt11528 cells treated with neomycin, or Pf101, but not in plants infiltrated with NPS3121 or water. These results suggest that there are unique biochemical events associated with the expression of a hypersensitive reaction which are distinct from other plant defense responses such as phytoalexin biosynthesis. In addition, our results support the hypothesis that there is a general, nonspecific mechanism for the induction of defense transcripts and phytoalexins by pathogenic and saprophytic bacteria that is distinct from the more specific mechanism associated with the induction of the hypersensitive reaction.

Journal Article↗

The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean. Determination of the structure by a combination of matrix-assisted laser desorption/ionization mass spectrometry and selective enzymic degradation.

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110-kDa glycoprotein related to the mammalian purple acid phosphatases with a two-metal cluster at the active site contains five oligosaccharide side chains/monomer. The N-linked glycan structures were characterized by selective enzymic degradation in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The purified protein was cleaved by cyanogen bromide. One 30-kDa large methionine-free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an alpha 1-3 fucose attached to the asparagine-linked N-acetylglucosamine prevented removal of the glycan by peptide N-glycosidase F; peptide N-glycosidase A removed all carbohydrates from the peptides. To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex-type xylose-containing structure with four of the five having an additional fucose.

Acid Phosphatase↗

Differences in expression between two seed lectin alleles obtained from normal and lectin-deficient beans are maintained in transgenic tobacco.

Using Agrobacterium-mediated transformation, two genes for phytohemagglutinin-L (PHA-L), the lectin seed protein of the common bean Phaseolus vulgaris, were stably integrated into the tobacco genome. The two alleles for PHA-L, dlec2 and pdlec2, were obtained from a normal cultivar (Greensleeves) and a lectin-deficient cultivar (Pinto) respectively. In the bean embryos, the expression of dlec2 is 30 times greater than the expression of pdlec2. In the dlec2-transformed tobacco, PHA-L accumulated specifically in the seeds at the same stages as the tobacco seed storage proteins and was degraded after germination. PHA-L was found in the embryo, and at a 5-7 times lower concentration in the endosperm tissue of the mature tobacco seeds. No PHA could be detected in other parts of the plants. We conclude that the signals for temporal and spatial regulation of the dlec2 gene are present in the DNA fragment used for transformation. Transformation with the second PHA-L allele pdlec2 from the cultivar Pinto caused the accumulation of about 50 times less PHA-L in tobacco seeds when compared to dlec2. We conclude from analyzing the 5' sequences of dlec2 and Pdlec2 that the low expression phenotype of the Pdlec2 allele could be due to the absence or mutation of a cis-acting element carried by the dlec2 fragment.

Journal Article↗

Nucleotide sequence of a bean (Phaseolus vulgaris) U1 small nuclear RNA gene: implications for plant pre-mRNA splicing.

We have isolated a U1 small nuclear RNA (snRNA) gene from common bean (Phaseolus vulgaris). The haploid bean genome contains only one or a few copies of the U1 snRNA gene. The bean and human U1 snRNA genes are 65% homologous but share no significant similarity in the 5' or 3' flanking regions. The predicted secondary structure of bean U1 snRNA is identical to that of other U1 snRNAs, and the sequences of the single-stranded regions have been highly conserved. Thus, both the sequence of the single-stranded regions and the secondary structure of U1 snRNA appear to be important for its function. The role of U1 snRNA in pre-mRNA splicing is probably similar in plants and animals.

Base Sequence↗

[Characterization of the nutritional potential of tepari bean (Phaseolus acutifolius) grown in Mexico].

The chemical, physical, antiphysiological and biological quality of the protein of three varieties (white, brown and black) of tepary bean (Phaseolus acutifolius) were studied. Its proximate composition was determined, as well as water absorption, hardness of the seed, tannin content, trypsin inhibitors and lectins. Beans were also submitted to differential scanning calorimetry of the starch. The biological quality of its proteins was also established, based on the protein efficiency ratio (PER) and protein apparent digestibility. It was found that the protein content of the bean was high (25%). The white variety had the highest water absorption and the lowest hardness of the seed when compared with the other varieties (p less than 0.05). The black variety had a lower starch gelatinization energy and a higher cooking time than the white and brown varieties. The tannin content of the three varieties was low, particularly in the white one. The trypsin inhibitor and lectin content were relatively high in the raw beans, but disappeared almost totally during cooking. The biological quality of the tepari proteins was higher than that of the common bean cultivated under the same conditions, More evidently so in the white bean (PER = 1.48 as compared to 1.23 in pinto bean). It can be concluded that the tepary bean is a potentially good protein source and has also a great potential ad donor of genes for the obtention of better bean varieties from the culinary, nutritional and toxicological points of view.

Analysis of Variance↗

Semienclosed Tube Cultures of Bean Plants (Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique.

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.

Journal Article↗

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

Journal Article↗

Bean ribonuclease-like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression.

The intracellular pathogenesis-related (PR) proteins of common bean (Phaseolus vulgaris L.) are encoded by a highly polymorphic family of at least 20 genes. One member, the Ypr10*c gene, has been isolated and characterised. The deduced amino acid sequence of the encoded protein, PR-10, exhibits similarities to tree-pollen allergens, to food allergens from celery and apple and to ginseng ribonuclease peptide sequences. We show by RNA blot analysis that the Ypr10 gene family, including Ypr10*c, is strongly expressed in bean roots. In leaves Ypr10 transcript levels are low in young and mature stages but are elevated during senescence and in diseased states. Dark treatment of leaves causes strong induction of Ypr10 transcripts, which is reversible by light, and diurnal rhythms of transcript accumulation during the night are observed. Ypr10 genes are responsive to external stimuli related to pathogen-defence such as glutathione or salicylic acid. Transcriptional activity of a Ypr10*c promoter-beta-glucuronidase fusion gene in transgenic tobacco was observed in roots, in developing xylem and phloem of stems, and in the blade of senescent leaves, with highest levels at the onset of senescence. The most striking characteristic of developmental expression was the specific localisation of beta-glucuronidase activity in the transmitting tract of styles in flowers at anthesis. Feeding of various pathogen-related and stress-related stimuli to young tobacco leaves led to accumulation of GUS activity in leaf blades. We identify considerable spatio-temporal similarities between reported expression patterns of Ypr10 genes and ribonuclease genes, which, together with the significant sequence similarity to the ginseng ribonuclease, support the hypothesis of a ribonuclease function for PR-10 proteins and allow the prediction of possible biological roles.

Allergens↗

The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing.

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(III)-Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)-Fe(II) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-C-generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.

Acid Phosphatase↗