Nitric oxide increases tolerance responses to moderate water deficit in leaves of Phaseolus vulgaris and Vigna unguiculata bean species

Drought stress is one of the most intensively studied and widespread constraints, and nitric oxide (NO) is a key signaling molecule involved in the mediation of abiotic stresses in plants. We demonstrated that a sprayed solution of NO from donor sodium nitroprusside increased drought stress tolerance responses in both sensitive (Phaseolus vulgaris) and tolerant (Vigna unguiculata) beans. In intact plants subjected to halting irrigation, NO increased the leaf relative water content and stomatal conductance in both species. After cutting leaf discs and washing them, NO induced increased electrolyte leakage, which was more evident in the tolerant species. These leaf discs were then subjected to different water deficits, simulating moderate and severe drought stress conditions through polyethylene glycol solutions. NO supplied at moderate drought stress revealed a reduced membrane injury index in sensitive species. In hydrated discs and at this level of water deficit, NO increased the electron transport rate in both species, and a reduction of these rates was observed at severe stress levels. Taken together, it can be shown that NO has an effective role in ameliorating drought stress effects, activating tolerance responses at moderate water deficit levels and in both bean species which present differential drought tolerance.     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.     

Expression of a-Amylase in Phaseolus vulgaris and Vigna mungo Plants

Levels of starch and soluble sugar in pods of Phaseolus vulgarisand Vigna mungo plants were analyzed during the course of maturationof fruits. The results suggest that the immature pods of P.vulgaris function to some extent as temporary reservoirs ofcarbohydrates for growth of seeds. A less clear pattern of accumulationof starch was observed in pods of maturing fruits of Vigna mungo.Measurements of a-amylase activites in pods of maturing fruitsand immunoblotting with an antiserum against     

Isoenzymes of Superoxide Dismutase in Nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp

The activity and isozymic composition of superoxide dismutase (SOD; EC 1.15.1.1) were determined in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. formed by Rhizobium phaseoll 3622, R. Ieguminosarum 3855, and Bradyrhizobium sp. BR7301, respectively. A Mn-SOD was present in Rhizobium and two in Bradyrhizobium and bacteroids. Nodule mitochondria from all three legume species had a single Mn-SOD with similar relative mobility, whereas the cytosol contained several CuZn-SODs: two in Phaseolus and Pisum, and four in Vigna. In the cytoplasm of V. unguiculata nodules, a Fe-containing SOD was also present, with an electrophoretic mobility between those of CuZn- and Mn-SODs, and an estimated molecular weight of 57,000. Total SOD activity of the soluble fraction of host cells, expressed on a nodule fresh weight basis, exceeded markedly that of bacteroids. Likewise, specific SOD activities of free-living bacteria were superior or equal to those of their symbiotic forms. Soluble extracts of bacteria and bacteroids did not show peroxidase activity (EC 1.11.1.7), but the nodule cell cytoplasm contained diverse peroxidase isozymes which were readily distinguishable from leghemoglobin components by electrophoresis. Data indicated that peroxidases and leghemoglobins did not significantly interfere with SOD localization on gels. Treatment with chloroform-ethanol scarcely affected the isozymic pattern of SODs and peroxidases, and had limited success in the removal of leghemoglobin.     

Use of intrinsic antibiotic resistance for characterisation and identification of rhizobia from nodules of Vigna unguiculata (L) Walp. and Phaseolus vulgaris (L)

Intrinsic resistance to low concentrations of antibiotics was used to characterise 83 isolates from nodules of cowpea (Vigna unguiculata) and field bean (Phaseolus vulgaris). Characterisation and differentiation of isolates from cowpea was made difficult by associated fast-growing bacteria inside the nodule tissue. Thus, reliable pure culture was difficult to secure without repeated isolation and even via nodulation of the appropriate homologous host. Although the technique may be satisfactory for differentiation and identification of fast-growing rhizobia, it is rated inferior to serology on aspects of facility, time and accuracy where rhizobia from cowpea nodules are concerned. Fingerprint patterns of isolates revealed considerable heterogeneity amongst the populations even where there was commonality of location and/or host plant. Pure cultures of slow-growing rhizobia from V. unguiculata nodules were generally more resistant to the concentrations of antibiotics used than fast-growing nodule bacteria from P. vulgaris.     

Compensation among root classes in Phaseolus vulgaris L.

The activity of soil pathogens, competition for assimilates, and the changing availability of below-ground resources make root systems subject to a continuous and dynamic process of formation and loss of both fine and coarse roots. As hypocotyl borne roots appear later than other root classes, they may serve to functionally replace basal and primary roots lost to biotic and abiotic stress. Using common bean (Phaseolus vulgaris L.), we conducted experiments in solution and solid media culture with treatments involving the removal of part of the root system (basal, hypocotyl borne or primary roots), phosphorus availability, and depth of seeding to test the hypothesis that there are compensation mechanisms among basal, hypocotyl borne and primary roots to cope with the loss of part of the root system. The root system was highly plastic in response to root excision, which resulted in the maintenance of below-ground biomass accumulation. In most cases, this compensation among root classes was enough to maintain plant performance in both phosphorus sufficient and phosphorus stressed plants. Removal of a specific root class induced an increase in the growth of the remaining root classes. All root classes, but especially the primary root, contributed to the compensation mechanism in some way. Primary roots represented around 10% of the root system in control plants and this proportion increased dramatically (up to 50%) when other root classes were removed. In contrast, negligible compensatory re-growth was observed following removal of the primary root. Greater planting depth increased the production of hypocotyl borne roots at the expense of basal roots. The proportion of hypocotyl borne roots increased from 25% of the whole root system when seeds were placed at a depth of 2 cm to 30% when they were placed at 5 cm and to 38% when placed at 8 cm, with corresponding decreases in the proportion represented by basal roots. The common feature of our observations is the innate ability of the root system for its own regeneration. Total root biomass maintained strict allometric relationships with total shoot biomass in all treatments. Re-stabilization of root to shoot balance after partial root loss is governed by overall plant size following allometric relationships similar to undisturbed plants. However, the pattern of this root regeneration was not uniform since the way the three root classes compensated each other after the removal of any one of them varied among the different growth media and phosphorus supply conditions. The resulting changes in root architecture could have functional significance for soil resource acquisition.     

Chlorophyll a Fluorescence Analysis in Response to Excitation Irradiance in Bean Plants (Phaseolus vulgaris L. and Vigna unguiculata L. Walp) Submitted to High Temperature Stress

Bean plants Phaseolus vulgaris L. (cv. Carioca and Negro Huasteco) and Vigna unguiculata L. Walp (cv. Epace-10) were grown in a growth chamber with a photosynthetic photon flux density of 200 mol m^–2 s^–1 at leaf level and air temperature of 25+1 °C. Fully expanded, first pair leaves of 12-d-old plants were submitted for 90 min to high temperature (25, 30, 35, 40, 45, and 48 °C). Chlorophyll a fluorescence parameters (ETR, q_P, q_N, and F₀) were investigated using a modulated fluorimeter at 25 °C during recovery considered here as 48 h after stress induction period. An accentuated decrease in q_P and an increase in q_N at 48 °C in Carioca and Negro Huasteco was not observed in Epace-10. In response to excitation irradiance a great potential for ETR was found in Negro Huasteco at 25 °C, also demonstrated by net photosynthetic rate. At 48 °C ETR was high for Epace-10 while it was equal to zero for Carioca and Negro Huasteco. Tolerance to high temperature observed in Epace-10 provided important information about the adaptative characteristics of Vigna cultivars to warm climates.     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Efficiency of three PCR-based markers in assessing genetic variation among cowpea (Vigna unguiculata subsp. unguiculata) landraces.

The main objective of this study was to investigate the efficiency of RAPD, AFLP, and SAMPL marker systems in detecting genetic polymorphism in cowpea landraces (Vigna unguiculata subsp. unguiculata (L.) Walp.) that probably share a similar genetic pool. A second objective was to determine the level of diversity among landraces from a restricted area, to define the most appropriate strategy of on-farm conservation. Each marker system was able to discriminate among the materials analysed, but a clear distinction between all the local varieties was only obtained with AFLP and SAMPL markers. The average diversity index was quite similar for each marker system, but owing to the differences in the effective multiplex ratio values the marker index was higher for the AFLP and SAMPL systems than for the RAPD system. The AFLP and SAMPL techniques appear to be more useful than the RAPD technique in the analysis of limited genetic diversity among the cowpea landraces tested. The significant correlations of SAMPL similarity and cophenetic matrices with those of the other markers, and the lower number of primer combinations required, indicate that this technique is the most valuable. The low genetic similarity detected among landraces suggests that all the cowpea landraces should be maintained on the respective farms from which they came.     

Comparative molecular mapping in Ceratotropis species using an interspecific cross between azuki bean (Vigna angularis) and rice bean (V. umbellata)

A genetic linkage map was developed with 86 F₂ plants derived from an interspecific cross between azuki bean (Vigna angularis, 2n=2x=22) and rice bean (V. umbellata, 2n=2x=22). In total, 14 linkage groups, each containing more than 4 markers, were constructed with one phenotypic, 114 RFLP and 74 RAPD markers. The total map size was 1702 cM, and the average distance between markers was 9.7 cM. The loci showing significant deviation from the expected ratio clustered in several linkage groups. Most of the skewed loci were due to the predominance of rice bean alleles. The azuki-rice bean linkage map was compared with other available maps of Vigna species in subgenus Ceratotropis. Based on the lineage of the common mapped markers, 7 and 16 conserved linkage blocks were found in the interspecific map of azuki bean ×V. nakashimae and mungbean map, respectively. Although the present map is not fully saturated, it may facilitate gene tagging, QTL mapping and further useful gene transfer for azuki bean breeding. Received: 20 March 1999 / Accepted: 29 April 1999     

Subgroups of the Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum

Rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group. To delineate the degree of specificity exhibited by four legumes in the cowpea miscellany, we tested the symbiotic characteristics of indigenous cowpea bradyrhizobia on cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea). The most-probable-number counts of indigenous bradyrhizobia at three sites on Maui, Hawaii, were substantially different on the four hosts: highest on siratro, intermediate on cowpea, and significantly lower on both lima bean and peanut. Bradyrhizobia from single cowpea nodules from the most-probable-number assays were inoculated onto the four hosts. Effectiveness patterns of these rhizobia on cowpea followed a normal distribution but were strikingly different on the other legumes. The effectiveness profiles on siratro and cowpea were similar but not identical. The indigenous cowpea-derived bradyrhizobia were of only moderate effectiveness on siratro and were in all cases lower than the inoculant-quality reference strain. Between 5 and 51% of the bradyrhizobia, depending on site, failed to nodulate peanut, whereas 0 to 32% failed to nodulate lima bean. No significant correlation was observed between the relative effectiveness of the bradyrhizobia on cowpea and their corresponding effectiveness on either lima bean or peanut. At all sites, bradyrhizobia that were ineffective on cowpea but that effectively nodulated lima bean, peanut, or both were found. Eighteen percent or fewer of the bradyrhizobia were as effective on lima bean as the reference inoculant strain; 44% or fewer were as effective on peanut as the reference strain. Only 18% of all cowpea-derived bradyrhizobia tested were able to form N(2)-fixing nodules on both lima bean and peanut. These results indicate the need to measure indigenous bradyrhizobial population characteristics directly with the crop of interest to obtain an accurate assessment of the need to inoculate.     

Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.)

Asparagus bean (V. unguiculata ssp. sesquipedialis), a specific form of cowpea (V. unguiculata L. Walp.), is cultivated as a vegetable crop throughout eastern and southern Asia for its tender long pods. Little is known about the genetic relationship between asparagus bean and the broader species, particularly the dominant ssp. unguiculata. We report here the development and transferability of simple sequence repeat (SSR) markers, over 40% of which are EST-derived, from ssp. unguiculata to asparagus bean and the use of a subset of the polymorphic markers to assess the genetic diversity of asparagus bean cultivars from diverse geographic origins across China. A total of 410 EST derived SSR (eSSR) markers and 600 SSR markers derived from cowpea genespace sequences (GSS) were developed, with a cross-subspecies transferability of 100% and 98.5%, respectively. In a recombinant inbred line population of asparagus bean, a 1:1 segregation was observed for most loci. Principal coordinate analysis (PCA) and phylogenetic clustering based on 62 alleles detected by 14 polymorphic SSR markers distinguished ssp. unguiculata and sesquipedialis into separate groups. Improved asparagus bean cultivars in China generally have a narrow genetic basis compared with landraces varieties. This suggests that asparagus bean breeding programs need to consider utilizing landrace germplasm to enhance genetic variability and ensure long-term gains from selection and reduce genetic vulnerability to pathogen/pest epidemics. Because of their transferability across subspecies, the SSR markers described in this study could be effectively employed in cross-subspecies trait introgression breeding from ssp. unguiculata to sesquipedialis.     

Callus induction from protoplasts of V. unguiculata,V. sublobata and V. mungo

Summary Protoplasts were isolated from hypocotyl of V. mungo (L.) Hepper or hypocotyl-derived callus of V. sublobata (Phaseolus sublobata Roxb.) and V. unguiculata (L.) Walp (syn. V. sinensis (L.) Saviex Hassk) using an enzyme solution comprising Cellulase 2.5%, Macerozyme, Hemicellulase and Driselase each at a 0.5% level in 0.5 M sorbitol. Isolated protoplasts were cultured in Murashige and Skoog's (1962) basal liquid medium supplemented with BA, NAA, 2,4-D (1 mg/l each) and sucrose (14%). After four weeks, protoplast colonies were transferred to the same medium with a reduced level of sucrose (7%). Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones of V. unguiculata differentiated roots on auxin/cytokinin supplemented media. Alternative methods for shoot differentiation from protoplastderived cultures were tried by the use of Agrobacterium tumefaciens shooter strains pGV 2215 or pGV 2298 or wild type strain B6S3.     

QTL mapping for nodule number and common bacterial blight in Phaseolus vulgaris L.

A recently developed bean RFLP linkage map was used to identify genetic elements affecting quantitative trait loci (QTLs) in two contrasting common bean genotypes, BAT-93 and Jalo EEP558, under two levels of mineral nitrogen: low – 0.25 mM NH4NO3 and a high – 6 mM NH4NO3. QTLs affecting nodule number (NN) and response to Xanthomonas campestris bv. phaseoli, which causes common bacterial blight (CBB) were identified and mapped. Analyses of 70 F2-derived F3 families, using the F1, the two parents, and a nodulation-defective mutant (Nod-) inoculated with R. tropici UM1899 under both levels of N showed significant differences (P#60;0.0001) among the F3 families for NN.Under low N, three genomic regions influenced both traits, with seven linked markers. In three of the six regions influencing NN, higher NN was associated with the Jalo EEP-558 allele, whereas in only two regions was the BAT-93 allele associated with higher NN. One-way analysis of variance, with each marker as the independent variable and NN as the dependent variable, and interval mapping analysis identified four QTLs, which accounted for 45% of the total variation, and two additional QTLs near to yet unassigned loci. In linkage group D7, one QTL mapped to the same region as a QTL for CBB.Under high N, three additional regions were linked to NN, one where the BAT-93 allele was closely associated with CH18 (chitinase), and the others where the Jalo EEP-558 allele was associated with CHS (chalcone synthetase) and PAL-1 (phenylalanine ammonia lyase). Four regions for CBB were mapped adjacent to or in the same region as a QTL for NN. Thus, N showed dual and opposite effects on the expression of NN and CBB. Analysis of these RFLP markers revealed these hidden favorable alleles and can serve as an indirect selection tool to increase NN and resistance to CBB.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: I. Occurrence of thiol proteinase inhibitors in plants and purification from Vigna unguiculata subsp. cylindrica

Inhibitors of the thiol proteinase, papain (EC 3.4.22.2), were shown to be present in 11 species of 10 genera of plants. The inhibitor activity was nondialyzable, and precipitated by ammonium sulfate. Tissue cultures from a number of plant genera consisting of rapidly dividing cells contained latent papain inhibitor that could be activated upon heating. Four isoinhibitors of plant thiol proteinases from seeds of the legume Vigna unguiculata subsp. cyclindrica were purified to apparent homogeneity by acrylamide gel electrophoresis with or without sodium dodecyl sulfate. The inhibitors were present in very small amounts compared to the trypsin inhibitors and the degree of purification of the homogeneous isoinhibitors on the assumption that all were present initially in equal amounts was 15,000- to 60,000-fold. The isoinhibitors did not inhibit pepsin, bromelain, and the serine proteinases, trypsin, chymotrypsin, and subtilisin. They were specific for papain, chymopapain, and ficin but their inhibition of the proteinase, esterase, and amidase activities of the three enzymes differed.     

Molecular characterization of intergenic spacer region of 5S ribosomal RNA genes in subgenus Vigna: extensive hybridization among V. unguiculata subspecies

We report the molecular analysis of the 5S ribosomal RNA intergenic spacer (IGS) region from 57 Vigna species of subgenus Vigna. Sequence analysis revealed that the 5S IGS was highly variable in length (189?C237?bp) and sequence (58% polymorphic sites). Most of the Vigna species analysed harboured a single type of 5S rRNA repeat unit, except V. unguiculata and V. reticulata that showed multiple ??intragenomic?? 5S types. The intragenomic 5S types among the six V. unguiculata subspecies were characterized by PCR-RFLP, genomic RFLP and sequencing. The 5S IGS was phylogenetically informative (comparable to ITS-1 and ITS-2 spacers) in inferring species relationships among the Vigna species analysed. However, due to the presence of multiple intragenomic 5S types and their incomplete homogenization among V. unguiculata subspecies the relationships in section Catiang could not be resolved below species level. The results presented indicate that intraspecies hybridization might have resulted in the ??horizontal transfer?? of 5S types among the V. unguiculata subspecies, while their maintenance could be due to a slow molecular drive.     

Genetic relationships among subspecies of Vigna unguiculata (L.) Walp. based on allozyme variation

 The cowpea [Vigna unguiculata (L.) Walp.] is a morphologically and genetically variable species composed of wild perennial, wild annual, and cultivated forms that are mainly used for edible seeds and pods. In this study, genetic variation in 199 germplasm accessions of wild and cultivated cowpea was evaluated using an allozyme analysis. The results from this survey showed that wild cowpea exhibits genetic variation perfectly fitted with the existing morphological classification. The cowpea gene-pool is characterized by its unusually large size. It encompasses taxa (ranked as subspecies) that could be considered as different species considering the high genetic distances observed between accessions belonging to different taxa. These subspecies can be classified into three groups characterized by their breeding systems: perennial outcrossers, perennial out-inbreds, and inbred annuals. Allozyme data confirm this grouping. Perennial outcrossers look primitive and are more remote from each other and from perennial out-inbreds. Within this large gene-pool, mainly made of perennial taxa, cultivated cowpeas (ssp. unguiculata var. unguiculata) form a genetically coherent group and are closely related to annual cowpeas (ssp. unguiculata var. spontanea) which may include the most likely progenitor of cultivated cowpeas. Received: 15 June 1998 / Accepted: 29 September 1998