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A putative precursor protein in the evolution of the bean alpha-amylase inhibitor.

Seeds of the common bean Phaseolus vulgaris and the tepary bean (P. acutifolius) contain a family of plant defence proteins that includes phytohaemagglutinin (PHA), arcelin and alpha-amylase inhibitor (alpha AI). These homologous proteins differ by the absence of short loops at the surface of the protein and by the presence of a proteolytic processing site (Asn77) that allows alpha AI to be post-translationally cleaved and activated. We now report the derived amino acid sequence of two amylase inhibitor-like (AIL) proteins that are not proteolytically processed, although they have the typical processing site. One protein is from the common bean, and the other from the tepary bean. On a dendrogram, these proteins are grouped with alpha AIs rather than with the arcelins or lectins. alpha AI differs from AIL primarily by the deletion of a 15-amino-acid segment from the middle of the AIL sequence. When alpha AI is expressed in tobacco, it is proteolytically processed to form an active molecule. However, AIL sequences are not processed. We suggest that the AIL proteins may be an intermediate in the evolution of an active alpha AI.

Amino Acid Sequence↗

Foliar Chlorosis in Symbiotic Host and Nonhost Plants Induced by Rhizobium tropici Type B Strains.

Rhizobium tropici CIAT899 induced chlorosis in the leaves of its symbiotic hosts, common bean (Phaseolus vulgaris L.), siratro (Macroptilium atropurpureum Urb.), and Leucaena leucocephala (Lam.) de Wit. Chlorosis induction by strains CIAT899 and CT9005, an exopolysaccharide-deficient mutant of CIAT899, required carbon substrate. When the bacteria were added at planting in a solution of mannitol (50 g/liter), as few as 10 cells of CIAT899 were sufficient to induce chlorosis in bean plants. All carbon sources tested, including organic acids and mono- and disaccharides, supported chlorosis induction. The addition of a carbon source did not affect the growth rate or the population density of CT9005 in the bean plant rhizosphere. Cell-free filtrates of cultures of CT9005 did not induce detectable chlorosis. All type B strains of R. tropici tested also induced chlorosis in common bean. Type A strains of R. tropici and all other species of bacteria tested did not induce chlorosis. Several lines of evidence indicated that nodulation was not required for chlorosis induction. Strain RSP900, a pSym-cured derivative of CIAT899, induced chlorosis in wild-type P. vulgaris. In addition, NOD125, a nodulation-defective line of common bean, developed chlorosis when inoculated with CIAT899, but did not develop nodules. CIAT899 consistently induced severe chlorosis in the leaves of the nonhost legumes alfalfa (Medicago sativa L.) and Berseem clover (Trifolium alexandrinum L.), and induced chlorosis in 29 to 58% of the plants tested of sunflower, cucumber, and tomato seedlings, but it did not induce chlorosis in the leaves of corn or wheat. Chlorosis induction in nonhost plants also required carbon substrate. The data are consistent with the hypothesis that R. tropici type B strains produce a chlorosis-inducing factor that affects a wide range of plant species.

Journal Article↗

Purification and characterization of two alpha-amylase inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray).

Two proteinaceous alpha-amylase inhibitors termed alphaAI-Pa1 and alphaAI-Pa2 were purified from seeds of a cultivated tepary bean (Phaseolus acutifolius A. Gray, cv. PI311897). The two inhibitors differed in their specificity towards alpha-amylases of insect pests such as bruchids, although neither showed any inhibitory activity against alpha-amylases of mammalian, bacterial or fungal origin. AlphaAI-Pa2 resembles two common bean inhibitors, alphaAI-1 and alphaAI-2, in several characteristics such as N-terminal amino acid sequences and oligomeric structure being composed of alpha and beta subunits. In contrast alphaAI-Pa1 is composed of a single glycopolypeptide with a molecular mass of 35 kDa, and its N-terminal amino acid sequence resembled that of seed lectins in tepary bean and common bean. The information on the two tepary bean alpha-amylase inhibitors may be useful not only for providing insight into critical structure for the specificity towards different alpha-amylase enzymes but also for enhancing insect resistance in crops.

Amino Acid Sequence↗

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen.

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.

Amino Acid Sequence↗

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies.

Base Sequence↗

Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses.

The 3'-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3'-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed.

Amino Acid Sequence↗

Lectin and lectin-related proteins in lima bean (Phaseolus lunatus L.) seeds: biochemical and evolutionary studies.

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.

Amino Acid Sequence↗

Analysis of Rhizobium etli and of its symbiosis with wild Phaseolus vulgaris supports coevolution in centers of host diversification.

Common beans (Phaseolus vulgaris) comprise three major geographic genetic pools, one in Mexico, Central America, and Colombia, another in the southern Andes, and a third in Ecuador and northern Peru. Species Rhizobium etli is the predominant rhizobia found symbiotically associated with beans in the Americas. We have found polymorphism in the common nodulation gene nodC among R. etli strains from a wide range of geographical origins, which disclosed three nodC types. The different nodC alleles in American strains show varying predominance in their regional distributions in correlation with the centers of bean genetic diversification (BD centers). By cross-inoculating wild common beans from the three BD centers with soils from Mexico, Ecuador, Bolivia, and Northwestern Argentina, the R. etli populations from nodules originated from Mexican soil again showed allele predominance that was opposite to those originated from Bolivian and Argentinean soil, whereas populations from Ecuadorian soil were intermediate. These results also indicated that the preferential nodulation of beans by geographically related R. etli lineages was independent of the nodulating environment. Coinoculation of wild common beans from each of the three BD centers with an equicellular mixture of R. etli strains representative of the Mesoamerican and southern Andean lineages revealed a host-dependent distinct competitiveness: beans from the Mesoamerican genetic pool were almost exclusively nodulated by strains from their host region, whereas nodules of beans from the southern Andes were largely occupied by the geographically cognate R. etli lineages. These results suggest coevolution in the centers of host genetic diversification.

Acyltransferases↗

Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco.

The storage protein phaseolin accumulates during seed development in protein bodies in cotyledons of the common bean Phaseolus vulgaris. Hall et al. (In L Van Vloten-Doting, TC Hall, eds, Molecular Form and Function of the Plant Genome, 1985 Plenum Press, In press) recently reported the expression of a gene coding for phaseolin and the accumulation of phaseolin protein in developing seeds of tobacco plants regenerated from transformed callus cells. The protein did not accumulate in other organs of the plants. Mature seeds from normal and transformed tobacco plants were obtained and the subcellular distribution of phaseolin in the seeds was examined using both light and electron microscopic immunocytochemical methods. Phaseolin was found in six of seven transformed tobacco embryos examined, but was present in only one endosperm of five. When present, phaseolin was located exclusively in the protein bodies of the embryonic and endospermic cells. Furthermore, phaseolin was restricted solely to the amorphous matrix of the protein bodies and was excluded from the globoid and proteinaceous crystalloid components of these organelles. The subcellular location of phaseolin in seeds from transformed tobacco plants is similar to that seen in mature seeds of the common bean indicating that in the transformed cells the protein is targeted to the right subcellular compartment.

Journal Article↗

Pollen fertility restoration by nuclear gene Fr in CMS bean: nuclear-directed alteration of a mitochondrial population.

Two nuclear genes, Fr and Fr2, have been identified that restore pollen fertility to cytoplasmic male sterile (CMS) common bean (Phaseolus vulgaris L.) by apparently distinct mechanisms. Whereas Fr2 appears to suppress the expression of a male sterility associated mitochondrial sequence (designated pvs), Fr restores pollen fertility by causing the elimination of this unusual mitochondrial DNA-segment. To further investigate the mechanism of Fr action, Fr and Fr2 were cointroduced into the nucleus of a bean line containing the sterility inducing cytoplasm. When the effect of pvs was suppressed by Fr2, the presence of Fr no longer directed the elimination of the mitochondrial pvs sequence. This result suggests that the Fr function is dependent on proper expression of the pvs sequence. To evaluate the temporal and spatial patterns of Fr action, we undertook a polymerase chain reaction-based approach to trace the fate of the pvs sequence in different tissues of F2 and F3 fertile-restored plants derived from a genetic cross between a cytoplasmic male sterile line of common bean, CMS-Sprite (frfr), and fertility restorer line R351 (FrFr). We demonstrate that the Fr-directed disappearance of pvs sequence occurs during flower development. Elimination of the pvs sequence from developing megaspores results in permanent fertility restoration in the following generations. Genetic analysis demonstrated that permanent fertility restoration, that is, the complete elimination of pvs from reproductive tissues requires two doses of the Fr allele or the absence of fr in F2 individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Antimutagenic effects of natural phenolic compounds in beans.

Polyphenols in fruits, vegetables (e.g., flavonols like quercetin) and tea (e.g., catechins such as epigallocatechin gallate) are good antioxidants with antimutagenic and anticarcinogenic properties. In the present study, the Salmonella typhimurium tester strain YG1024 was used in the plate-incorporation test to examine the antimutagenic effect of phenolic compounds, extracted from common beans (Phaseolus vulgaris), on 1-NP and B[a]P mutagenicity. Dose-response curves for 1-NP and B[a]P were obtained; the number of net revertants/plate at the peak mutagenic dosage were 880 for 1-NP and 490 for B[a]P. For the antimutagenicity studies doses of 0.1 microg/plate and 2 microg/plate for 1-NP and B[a]P, respectively, were chosen. We obtained a dose-response curve of ellagic acid (EA) against B[a]P and 1-NP mutagenicity. To test the bean extract, a dose of 300 microg/plate of EA was chosen as the antimutagenic control. The EA and bean extracts were not toxic to the bacteria at the concentrations tested. The inhibitory effects of the bean extracts and EA against B[a]P mutagenicity were dose-dependent. The percentages of inhibition produced against B[a]P (2 microg/plate) using 300 microg/plate of EA and for the extracts 500 microg equivalent catechin/plate were 82%, 83%, 81% and 83% for EA, water extract, water/methanol extract and methanol extract, respectively. However, for 1-NP mutagenicity, only the methanolic extract from beans showed an inhibitory effect. These results suggest that common beans, as other legumes, can function as health-promoting foods.

Antimutagenic Agents↗

GuaB activity is required in Rhizobium tropici during the early stages of nodulation of determinate nodules but is dispensable for the Sinorhizobium meliloti-alfalfa symbiotic interaction.

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate the timing of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.

Artificial Gene Fusion↗

Extensive modifications for methionine enhancement in the beta-barrels do not alter the structural stability of the bean seed storage protein phaseolin.

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. An Escherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded in E. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the beta-barrel and loop structures, with extremely small effects on protein stability.

Amino Acid Sequence↗

Characterization of polyphenolics in the seed coat of Black Jamapa bean (Phaseolus vulgaris L.).

The common bean contains phytochemicals, including phenolic compounds, which can provide health benefits to the consumer. Our objective was to characterize the polyphenolic compounds present in the seed coat of Black Jamapa bean and to test fractionation methods that permit the recovery of polyphenolics in their naturally occurring forms. A 100% methanol extract from seed coats was subjected to different chromatographic fractionation methods. Analysis by HPLC-MS revealed that a better separation of phytochemicals was achieved using direct silica gel fractionation, which allowed more accurate identification of compounds, especially of the flavonols. Anthocyanins, flavanol monomers, and heterogeneous flavanol oligomers up to hexamers were detected. To our knowledge, this is the first time that myricetin glycoside and proanthocyanidin oligomers containing (epi)-gallocatechin have been reported in the black bean. The fractionation methods used in this study produced large quantities of natural mixtures of flavonoids suitable for testing bioactivity and phytochemical interactions.

Anthocyanins↗

Effects of plant species, age and part on the disappearance of sevin, nuvacron and malathion residues.

Residues as determined by bioassay using Daphnia or mosquito larvae were in agreement with each other in most cases except sevin residues at 1 h and 8 days after treatment of mallow. The mosquito larvae failed to record nuvacron, sevin and malathion on 45-day-old plants on the 8th, 12th and 24th day, respectively, whereas residues on younger plants continued to affect mosquitoes up to the 12th day and disappeared only on the 24th day. Daphnia continued to show toxicity up to the 24th day on younger and older plants. Insecticide residues of nuvacron, malathion and sevin, found on the leaves 30- and 45-day-old plants of cotton, Jew's mallow and kidney beans after 1 h, 1, 4, 8, 12 and 24 days were estimated biologically by C. pipiens larvae and D. magna. Residues of insecticides disappeared more readily on bean pods than on bean leaves. Residues of sevin, malathion and nuvacron found on the pods 12 days after treatment as indicated by Daphnia were 0.189, 0.055 and 0.059 ppm respectively. They are far less than the corresponding residues on bean leaves. The 1-hour residue was higher on younger bean leaves than on mallow and cotton with very few exceptions (nuvacron, malathion and sevin: 2.125, 11.75 and 95 ppm on cotton leaves; 2.25 and 145 ppm on Jew's mallow and 3.750, 32.500 and 250 ppm on common bean leaves, respectively). These data were obtained with C. pipiens larvae. The picture was completely reversed on 45-day-old plants. 1-h deposits of malathion were higher on mallow than on cotton or beans (nuvacron, malathion and sevin; 2.3, 200 and 140 ppm on cotton leaves, 1.90, 191.15 and 92.86 ppm on mallow leaves, 2.25, 21.5 and 137.5 ppm on common bean leaves, respectively). These data were obtained with C. pipiens larvae. Nuvacron residues on 45-day-old mallow were less on mallow than on cotton or beans. Sevin was higher in 1-h residues on cotton and beans than on mallow. Mallow did not retain insecticides as long as did cotton and beans. The initial concentration of nuvacron was little less than that of sevin and malathion. It was more toxic to mosquito larva (LC50 = 0.0016 ppm) than malathion (LC50 = 0.0034 ppm) and sevin (LC50 = 0.075 ppm). Daphnia was more affected by malathion (LC50 = 0.000098 ppm) than by nuvacron (LC50 = 0.00024 ppm). Nuvacron was nearly equitoxic to sevin (LC50 = 0.00026 ppm) against Daphnia. In spite of this all tested plants, both young or older ones, retained nuvacron in the smallest quantities in proportion to the other insecticides. The 1-h residues were less on older plants (except cotton) for only nuvacron and sevin: 2 and 94.5, 1.9 and 92.8 ppm than on younger ones 2.4 and 137.2, 2.25 and 145 ppm as indicated by Daphnia and mosquito larvae respectively. This emphasizes that the initial concentration is not the limiting factor for the determination of the 1-h residue as the initial concentration was much higher on older than on younger plants.

Animals↗

The ecology and epidemiology of whitefly-transmitted viruses in Latin America.

Whitefly-transmitted geminiviruses are the most important constraint to the production of common bean and vegetable crops in the tropical lowlands and mid-altitude valleys of Latin America. Currently, over 30 distinct species of geminiviruses that are transmitted by the whitefly Bemisia tabaci infect common bean, tomato, pepper, cucurbits and other horticultural crops. A climate probability model (using FloraMap) was obtained using data from 304 geo-referenced locations where B. tabaci and geminiviruses cause significant damage. Clustering of the 304 points produced a simple model with two climatic variables: a dry season of at least 4 months each with less than 80 mm of rain and a mean temperature of the hottest month exceeding 21 degrees C. A modified Koeppen climate classification showed that 55% of the geminivirus-affected localities are in the tropical wet/dry region, 22% in the tropical and subtropical dry/humid climates and the remaining locations belonged to the wet equatorial and trade wind litoral climates. These findings contribute to understanding whitefly/geminivirus epidemics and implementation of sustainable integrated pest and disease management practices.

Animals↗

Pattern of fecal endogenous nitrogen excretion in rats fed leguminous diets.

The main objective of this investigation was to establish the pattern in relation to time of the rat fecal endogenous nitrogen excretion by continuous feeding of balanced diets containing common peas, cowpeas or common beans as the protein sources (10% protein), labeled with 1.000 atoms % of 15N-excess. Nitrogen of endogenous origin was measured by the isotope dilution method in a 6-day experiment. Fecal excretion of endogenous nitrogen of rats fed the leguminous diets was roughly twice that of rats fed the non-protein diet (88 mg, 42 mg), and the excretion of total fecal nitrogen did not differ among leguminous diets. From the third to the sixth day of the experiment, the endogenous nitrogen excretion, either as a percentage of quantity (mg), attained a statistically non different value (p > 0.05). A common pattern of excretion of fecal endogenous nitrogen as a function of time was expressed by a strong negative (r < -0.95) power regression (y = A.x-B) for the common pea, the cowpea or the common bean diets. Conversely, the excretion of dietary nitrogen did not show a common pattern as a function of time for all experimental diets.

Animals↗

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development.

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety "Red Kidney."

Journal Article↗