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PubMed2002

A novel subtilase from common bean leaves.

We describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.6, and SDS-PAGE in a single band corresponding to M(r) 72 kDa. The proteinase activity is maximal at pH 9.9 and shows high stability in the alkaline region. The relative activities of the proteinase for eight different synthetic substrates were determined. The requirement for Arg in the P1 position appeared obligatory. k(cat)/K(m) values indicate that, for highest catalytic efficiency, a basic amino acid is also required in the P2 position, presenting a motif typical of the cleavage site for the kexin family of subtilases. The sequence of the 17 N-terminal amino acids of this proteinase shows similarity to those of other plant subtilases, sharing the highest number of identical amino acids with proteinase C1 from soybean seedling cotyledons and a cucumisin-like proteinase from white gourd (Benincasa hispida).

Amino Acid Sequence↗

PubMed1990

Fertility Restoration Is Associated with Loss of a Portion of the Mitochondrial Genome in Cytoplasmic Male-Sterile Common Bean.

Restoration of pollen fertility to cytoplasmic male-sterile common bean by nuclear gene Fr is accompanied by mitochondrial (mt) DNA rearrangements within restored plants. These rearrangements are also observed upon spontaneous cytoplasmic reversion to fertility. An mtDNA fragment of at least 25 kilobases was lost from the genome upon restoration or reversion. This fragment contained DNA segments that were not repeated elsewhere in the genome and, therefore, were not detected within the genome upon fertility restoration. This result suggested that the particular mtDNA configuration absent from restored plants could not be maintained by a constant process of recombination but rather by autonomous replication. No evidence of excision of this region from the mt genome, in the form of a junction fragment associating flanking DNA regions, was detected in fertile restored plants. DNA gel blot hybridization of this mtDNA region, compared with hybridization to related regions of the mitochondrial genome that shared sequence homology, indicated that the mtDNA region associated with sterility was present in lower copy number. These observations, as well as the occurrence of similar or identical rearrangements upon spontaneous cytoplasmic reversion, indicate that the restoration of pollen fertility may be accompanied by loss of an independently replicating subgenomic DNA molecule from the mitochondrial genome.

Journal Article↗

PubMed1989

Heliotropic leaf movements in common beans controlled by air temperature.

Heliotropic leaf movements were examined in common beans (Phaseolus vulgaris cv Blue Lake Bush) under outdoor and laboratory conditions. Heliotropic leaf movements in well-watered plants were partly controlled by temperature, and appeared to be independent of atmospheric humidity and CO(2) concentration. When environmental conditions were held constant in the laboratory, increased air temperature caused bean leaves to orient more obliquely to a light source. Ambient CO(2), intercellular CO(2), and net photosynthesis were not correlated with the temperature-induced changes in heliotropic movements, nor did they significantly affect these movements directly. The effect of air temperature on leaf movements need not be mediated through a change in leaf water potential, transpiration, or leaf conductance. Air temperature modified laminar orientation in light through its effect on tissue temperature in the pulvinal region, not that of the lamina or petiole. However, under darkness the temperature effects on leaf movements were not expressed. Active heliotropic movements in response to air temperature allowed lamina temperature to remain close to the thermal optimum of photosynthesis. This temperature effect underlies a commonly observed pattern of leaf movements under well-watered conditions: a tendency for leaves to face the sun more obliquely on hot days than cool days.

Journal Article↗

PubMed2006

Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings.

Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119-1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation.

Cell Wall↗

PubMed1998

A cytoplasmic male sterility-associated mitochondrial peptide in common bean is post-translationally regulated.

Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.

Amino Acid Sequence↗

PubMedDate not supplied

Integration of simple sequence repeat (SSR) markers into a molecular linkage map of common bean (Phaseolus vulgaris L.).

Microsatellite or simple sequence repeat (SSR) markers have been successfully used for genomic mapping, DNA fingerprinting, and marker-assisted selection in many plant species. Here we report the first successful assignment of 15 SSR markers to the Phaseolus vulgaris molecular linkage map. A total of 37 SSR primer pairs were developed and tested for amplification and product-length polymorphism with BAT93 and Jalo EEP558, the parental lines of an F7 recombinant inbred (RI) population previously used for the construction of a common bean molecular linkage map. Sixteen of the SSRs polymorphic to the parental lines were analyzed for segregation and 15 of them were assigned to seven different linkage groups, indicating a widespread distribution throughout the bean genome. Map positions for genes coding for DNAJ-like protein, pathogenesis-related protein 3, plastid-located glutamine synthetase, endochitinase, sn-glycerol-3 phosphate acyltransferase, NADP-dependent malic enzyme, and protein kinase were determined for the first time. Addition of three SSR loci to linkage group B4 brought two separated smaller linkage groups together to form a larger linkage group. Analysis of allele segregation in the F7 RI population revealed that all 16 SSRs segregated in the expected 1:1 ratio. These SSR markers were stable and easy to assay by polymerase chain reaction (PCR). They should be useful markers for genetic mapping, genotype identification, and marker-assisted selection of common beans.

Alleles↗

PubMed1990

Inheritance of ambient ozone insensitivity in common bean (Phaseolus vulgaris L.).

The inheritance of ozone (O(3)) insensitivity in common bean (Phaseolus vulgaris L.) was evaluated using F(2) and F(3) populations under ambient conditions. This study was conducted over two growing seasons (1987, 1988) at Virginia State University, Randolph Research Farm, Petersburg, Virginia. Two populations were obtained by crossing insensitive plant introductions with sensitive commercial cultivars. Ratings on the scale of 1 to 5 (1 = 0 to 20% leaf injury, 2 = 21 to 40%, 3 = 41 to 60%, 4 = 61 to 80%, and 5 > 80%) were made on 160 F(2), F(3) progenies, and parental lines. Population mean injury ratings were recorded and estimates of genotypic, environmental, and phenotypic variances were computed. Estimates of heritability in the broadsense and of genetic advance were calculated for each population using F(2) and family component variance methods. Population means of the F(2) and F(3) progenies were not significantly different from their mid-parent values, suggesting that genetic variance was primarily additive. Broad-sense heritability estimates using F(2) variance method ranged from 51.4 to 70.5% and using family component variance method ranged from 62.1 to 75.6%. In this study, the computed genetic advance values closely parallel those of heritability estimated values. The high heritable nature of insensitivity would indicate that effective levels of insensitivity could be transferred to agronomically superior cultivars in a relatively short time.

Journal Article↗

PubMed2004

Analysis of hard-to-cook red and black common beans using Fourier transform infrared spectroscopy.

Extracted fractions from black and red common beans (Phaseolus vulgaris) were studied using Fourier transform infrared spectroscopy (FT-IR). Beans were stored under three conditions: control at 4 degrees C; hard-to-cook (HTC) at 29 degrees C, 65% RH for 3.5 months; and refrigerated at 2 degrees C, 79% RH for 3.5 months after a HTC period (called HTC-refrigerated). Two fractions isolated from the beans, the soluble pectin fraction (SPF) and the water insoluble residue of the cell wall (WIRCW), were analyzed using diffuse reflectance (DRIFTS) FT-IR. The soaking water and cooking water from the beans were also studied using attenuated total reflectance (ATR) FT-IR. The DRIFTS FT-IR results from the SPF and WIRCW fractions were consistent with previously published data for Carioca beans showing that in general, more phenolic compounds were associated with the SPF of HTC beans than in the control beans. Results also showed that HTC-refrigerated beans had higher concentrations of phenolic compounds than control beans in the SPF. The ATR FT-IR results for soaking and cooking waters from the HTC-refrigerated and HTC beans had higher concentrations of absorbing compounds than the control beans, indicating that they lost more constituents to the water. Additionally, results indicate that the mechanism(s) for reversibility of the HTC defect could be different than the one(s) involved in the development of the defect.

Chemical Phenomena↗

PubMed1998

clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

Amino Acid Sequence↗

PubMed1992

Serotype A and B strains of bean common mosaic virus are two distinct potyviruses.

The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.

Amino Acid Sequence↗

PubMed1993

Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses.

Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.

Journal Article↗

PubMed1999

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup.

Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4A1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3'-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3'-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups.

Amino Acid Sequence↗

PubMed1996

Phylogenetic relationships among Rhizobium species nodulating the common bean (Phaseolus vulgaris L.).

The phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16S ribosomal DNA genes of these organisms. The bean strains formed four separate clusters. One cluster was composed of Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and R. leguminosarum bv. phaseoli. Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain. Data for species identification were obtained from DNA-DNA reassociation experiments. The levels of DNA relatedness among strains belonging to the three biovars of R. leguminosarum ranged from 58 to 67%. The levels of DNA relatedness between R. leguminosarum bv. phaseoli and R. etli and R. tropici ranged from 43 to 45% and 13 to 16%, respectively. The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.

Base Sequence↗

PubMed1990

Correlations between Carbon Isotope Discrimination and Leaf Conductance to Water Vapor in Common Beans.

Carbon isotope discrimination (Delta) was measured in the field on 10 cultivars of common bean (Phaseolus vulgaris L.). There was substantial variation (more than 2 per thousand) in leaf Delta values and these differences were maintained between vegetative and reproductive developmental stages. These bean lines also exhibited substantial differences in leaf conductance to water vapor, and again these differences were maintained across developmental stages. The differences in leaf conductance were positively correlated with Delta values, whether conductance was measured as total leaf conductance or as the individual conductances of either upper or lower leaf surfaces. The observed differences in leaf conductance were not associated with differences in stomatal density. There were small differences among bean lines in their leaf Kjeldahl nitrogen contents, which is interpreted as indicating that photosynthetic capacity among bean lines was similar. Thus, because Delta values and leaf conductance were positively correlated, these data suggested that there may have been differences among bean lines in the extent to which stomata limited photosynthetic gas exchange rates.

Journal Article↗

PubMed1991

Alpha-amylase inhibitor, not phytohemagglutinin, explains resistance of common bean seeds to cowpea weevil.

There are claims that phytohemagglutinin (PHA), the lectin of common bean, Phaseolus vulgaris, is toxic when fed to the cowpea weevil, Callosobruchus maculatus, and that PHA serves as the chemical defense against this seed-feeding bruchid beetle (DH Janzen, HB Juster, IE Liener [1976] Science 192: 795-796; AMR Gatehouse, FM Dewey, J Dove, KA Fenton, A Pusztai [1984] J Sci Food Agric 35: 373-380). However, our studies indicate that neither PHA nor its isolectins have detrimental effects when fed to the cowpea weevil. To explain these contradictory results we characterized the commercial lectin source used by A. M. R. Gatehouse, F. M. Dewey, J. Dove, K. A. Fenton, A. Pusztai (1984, J Sci Food Agric 35: 373-380). We demonstrate here that the toxic effects of PHA to cowpea weevil are due to an alpha-amylase inhibitor contaminant in the commercial preparation.

Journal Article↗

PubMed1992

Competition among Rhizobium leguminosarum bv. phaseoli strains for nodulation of common bean.

Six effective Rhizobium leguminosarum bv. phaseoli strains were examined for nodulation competitiveness on common bean (Phaseolus vulgaris L.), using all possible two-strain combinations of inoculum. Nodule occupancy was determined with strain-specific fluorescent antibodies. The strains were divided into three groups according to their overall competitive abilities on pole bean cv. Kentucky Wonder and bush bean cv. Bountiful. Strains TAL 182 and TAL 1472 were highly competitive (greater than 70% nodule occupancy); strains KIM-5, Viking 1, and CIAT 899 were moderately competitive (approximately 50% nodule occupancy); and strain CIAT 632 was poorly competitive (less than 5% nodule occupancy). The competitiveness of the six strains was similar on the two host cultivars. The proportion of competing strains in the inoculum influenced the nodule occupancy of the highly competitive and moderately competitive strains, but not that of the poorly competitive strain. Two outstanding strains (TAL 182 and TAL 1472) were identified as ideal model strains for molecular and genetic studies on nodulation competitiveness.

Fabaceae↗

PubMed1988

Purification and characterization of arcelin seed protein from common bean.

Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M(r), deglycosylated M(r), and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.

Journal Article↗

PubMed2000

A GAL4-like protein is involved in the switch between biotrophic and necrotrophic phases of the infection process of Colletotrichum lindemuthianum on common bean. .

Random insertional mutagenesis was conducted with the hemibiotrophic fungus Colletotrichum lindemuthianum, causal agent of common bean anthracnose. Nine mutants that were altered in their infection process on the host plant were generated. One of these, H433 is a nonpathogenic mutant able to induce necrotic spots on infected leaves rapidly. These spots are similar to those observed during the hypersensitive reaction. Cytological observations showed that the development of the mutant H433 is stopped at the switch between the biotrophic and the necrotrophic phases. This mutant carries two independent insertions of the transforming plasmid pAN7-1. Complementation studies using the wild-type genomic regions corresponding to the two insertions showed that one is responsible for the H433 phenotype. Sequencing analysis identified a single open reading frame that encoded a putative transcriptional activator belonging to the fungal zinc cluster (Zn[II](2)Cys(6)) family. The corresponding gene was designated CLTA1 (for C. lindemuthianum transcriptional activator 1). Expression studies showed that CLTA1 is expressed in low amounts during in vitro culture. Targeted disrupted strains were generated, and they exhibited the same phenotype as the original mutant H433. Complementation of these disrupted strains by the CLTA1 gene led to full restoration of pathogenicity. This study demonstrates that CLTA1 is both a pathogenicity gene and a regulatory gene involved in the switch between biotrophy and necrotrophy of the infection process of a hemibiotrophic fungus.

Amino Acid Sequence↗