Purification and characterization of extracellular invertase from the hypocotyls of mung bean (Phaseolus radiatus L.)

The extracellular invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by ammonium sulfate fractionation and sequential chromatography over diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A-Sepharose 4B affinity and Sephadex G-200. The overall purification was about 77-fold with a recovery of about 11%. The finally purified enzyme exhibited a specific activity of about 113 μmol of glucose produced mg^-1 protein min^-1 at pH 5.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The enzyme had the native molecular mass of 134 kD and subunit molecular weight of 67 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of homodimeric proteins. On the other hand, the enzyme appeared to be a glycoprotein containing mannosyl residues on the basis of its ability to interact specifically with the immobilized Con A and the separability of invertase-Con A complex by methyl-α-D-mannopyranoside. The enzyme had a K_m for sucrose of 3.4 mM and its pH optimum of 4.0. The enzyme showed highest enzyme activity with sucrose as substrate. Raffinose and cellobiose were hydrolyzed at a low rate, maltose and lactose were not cleaved by the enzyme. These results indicate the extracellular invertase is a β-fructofuranosidase.     

甘油包埋绿豆SOD的稳定性研究

用不同浓度的甘油包埋绿豆(Phczeolus rodiatus L.)超氧化物岐化酶(Superoxide dismutase,SOD),对不同条件下制备的甘油SOD的稳定性进行评估.结果表明:甘油包埋绿豆SOD的稳定性受甘油浓度影响明显,12.5%～25%为最适甘油浓度.在10℃、pH 6.8、搅拌速度1000 r min-1.条件下包埋的甘油SOD在55℃的平均半衰期为25.1 d,为非包埋SOD的5.1倍.甘油浓度为25%,温度分别为5℃、10℃、25℃、45℃条件下,pH 3.8中包埋的绿豆SOD耐酸性较好,pH 8.9下包埋的绿豆SOD的抗碱能力较强.在包埋过程中添加一定浓度的Zn2 、Cu2 、Fe2 有利于其热稳定性的提高.甘油包埋SOD对一些常见的化妆品添加剂也有一定的抗性.由此可见,甘油包埋技术可望作为一项有应用前景的SOD活性保持新技术,有利于极端温度和极端pH条件下的SOD的应用.     

Ochratoxin-A production in Brazilian dry beans (Phaseolus vulgaris L.)

Ochratoxin A was produced, at concentrations of about 200 mg kg1 of dry beans (Phaseolus vulgaris L.) of each of five Brazilian commercial varieties. Both intact and decorticated kernels of the varieties Preto, Branco, Rosinha, Roxo and Carioca (22% moisture) were inoculated withAspergillus alutaceous and incubated at 25°C for 28 days. Results from thin-layer and column chromatography, mass, infrared, 1H-nuclear magnetic resonance and UV-spectrometry showed that 1) the common bean is a highly stimulatory substrate for the bioproduction of ochratoxin A and 2) the putative toxin extracted by the method of Soares & Rodriguez-Amaya was in fact ochratoxin A. Removal of the seed coat resulted in increased OTA production for all varieties, particularly for the Rosinha, Roxo and Carioca.     

seed rot and root rot complex of beans (Phaseolus vulgaris L.)

Fusarium solani (76%),Phyllosticta phaseolina (12%) andAspergillus flavus (10%) were isolated from bean(Phaseolus vulgaris) seeds showing lesions. Out of these the first two caused 30 per cent and 20 per cent seed rotting respectively after artificial seed inoculations. When inoculum was added into the soil before sowing the seed, 35 per cent and 10 per cent pre-emergence rot ocurred and there was 35 per cent and 15 per cent post-emergence blight when soil with healthy seedlings was inoculated withF. solani andP. phaseolina respectively.F. solani caused similar lesions on bean seeds when inoculated after an injury.F. oxysporum was isolated from the roots of dried bean plants showing necrosis.     

Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme

Five kinds of spacer arm attached chitosan hybrid hydrogels were tested for the possibility of being used as carriers for the immobilization of hydroperoxide lyase from amaranthus tricolor leaves. The 1,6-hexamethylenediamine attached chitosan-κ-carrageenan with biomimetic hydrophobic surface was proved to be the most suitable carrier. A maximum activity of 7.49 ± 0.19 U/g and a yield of 95% were obtained under optimized coupling condition. Meanwhile, the affinity between enzyme and substrates was not reduced after immobilization, as evidenced by the fact that the K_m value of hydroperoxide lyase decreased from 108.6 to 79.97 μM for 13-hydroperoxy-linoleic-acid and almost unchanged for 13-hydroperoxy-linolenic-acid. Furthermore, the thermal, operational and storage stabilities of HPL were significantly improved after immobilization. Using the immobilized enzyme as the catalyst, the yield of 2(E)-hexenal and hexanal reached 1374.8 ± 51.8 mg/L and 1987.9 ± 67.9 mg/L, respectively, and the amount of immobilized enzyme needed in the reaction mixture was much lower than its free counterpart.     

Light-dependent cytochrome P-450 changes in mung beans (Phaseolus aureus).

Maximum concentrations of microsomal cytochrome P-450 are present in 3-4 day-old mung beans (Phaseolus aureus). On illumination of dark-grown seedlings, cytochrome P-450 and later cytochrome P-450 undergo a rapid decrease in concentration in vivo, with an apparent half-time of about 6 h. Conversely light-grown seedlings, transferred to darkness, show a slow accumulation of cytochrome P-450, doubling time of about 30 h, with a later accumulation of cytochrome P-420. Microsomal cytochromes b559, b560.5 and b562.5 do not significantly alter on light-dark transitions. Possible functions for dark-induced cytochrome P-450 are discussed.     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

Fixed effect genetic analysis of a diallel cross in dry beans (Phaseolus vulgaris L.)

Summary A full diallel cross among four diverse homozygous strains of dry edible beans (Phaseolus vulgaris L.) was evaluated for yield, protein content, and culinary quality traits in the F₂ and F₃ generations in two locations. Interpretation of diallel effects [Method 1 Model I] using a fixed-effect genetic model made it possible to combine data from two generations into a single analysis and quantify the relative contributions of additive and dominance genetic effects to general (GCA) and specific (SCA) combining abilities. GCA was found to arise from three potential sources: additive effects, dominance interactions at homozygous loci, and average dominance interactions in hybrids involving the parent in question. SCA was found to be a function solely of dominance. Additive effects were the primary determinant of GCA and were highly significant. Specific dominance interactions were significant for seed yield, cooked bean moisture content, and texture but not for protein content. Texture was the only trait for which the additive-dominance model failed to provide an adequate fit to the data, suggesting that texture is significantly affected by epistatic interaction. One cross (Brazil-2 × Sanilac) was identified that exhibited a large heterotic effect for seed yield although the parents' additive effects were nonsignificant. Such a nicking effect was attributed to complementation between the two parents.Cooperative investigations of the Agricultural Research Service, U.S. Department of Agriculture and Michigan State University, East Lansing, MI 48824. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree at Michigan State University. Approved for publication by the Michigan Agricultural Experiment Station as Journal Article No. 11791. Research supported by USAID under a Title XII Bean/Cowpea CRSP and cooperative with Washington State University, Pullman, WA99164     

Nitrate reductase and nitrogenase activities of common beans (Phaseolus vulgaris L.) from different geographic locations

Summary Cultivars ofPhaseolus vulgaris (L.) from contrasting geographic locations were cultivated under fields conditions for measurements of nitrogenase and nitrate reductase activities. A first trial with two cultivars indicated that a tropical cultivar B-789 has a higher nitrogenase activity than a temperate one Elsa. And the converse was true for the nitrate reductase activity. While where a post flowering application was made, a renewal of nitrate reductase activity occurred. Further similar comparisons of both enzymatic activities upon eight tropical and temperate cultivars of equivalent vegetative cycles indicated, on the average, that tropical cultivars have a higher level of (C₂H₂) reduction and a lower nitrate reductase activity than temperature cultivars. These observations suggest that there exists an inverse relationship between the two enzymatic activities in common beans, and there probably exists genetic variability for a possible improvement of N-fixation ability. An early application of N-fertiliser upon the Elsa and B-789 plots promotes later nitrogenase activity while a post flowering application shows obvious a renewal of nitrate reductase activity. Thus, analyses of nitrate reductase and nitrogenase activities of a common bean crop at different physiological stages may give us an indication of the best time to apply supplementary nitrogen fertilisation to common beans to increase seed yield.     

The wild ancestors of urid and mung beans (Vigna mungo (L.) Hepper and V. radiata (L.) Wilczek)

The crop plants Vigna mungo (urid, urd or black gram) and V. radiata (mung bean or green gram: and related taxa have been studied by seed protein electrophoresis, leaf phenolics chromatography, vegetative morphology, and seed testa patterns. The results disprove the theory that these species are very closely related and have evolved from a single wild taxon. The present evidence shows that V. mungo var. mungo and I. radiata var. radiata have independent lineages and were domesticated from two very distinct taxa. namely V. mungo var. silvestris and V. radoata var. sublobata respectively.     

Iron bioavailability of common beans (Phaseolus vulgaris L.) intrinsically labeled with 59Fe

A radiobioassay was performed in rats with or without iron depletion to evaluate the iron bioavailability of diets enriched with common beans and with “multimixture”, a nutritional supplement based on parts of foods that are not usually eaten. The full-body ⁵⁹Fe level was determined after 5 h, the absorbed ⁵⁹Fe level was determined after 48 h, and the amount of ⁵⁹Fe retained was determined after 7 days. Iron bioavailability was assessed by the full-body radioactivity of the animals, determined using a solid scintillation detector. The iron bioavailability of common beans was higher in the iron-depleted animals (55.7%) than in the non-depleted animals (25.12%) because of the higher absorption rate in the iron-depleted animals. The multimixture did not influence dietary iron bioavailability. In addition, the iron bioavailability of common beans was similar to that observed in the standard source of iron for Wistar rats. Hence, common beans may be considered an adequate dietary iron source because of its high bioavailability.     

Heterosis for chiasma frequency and quantitative traits in common beans (Phaseolus vulgaris L.)

Summary Ten genotypes, including inbreds, hybrids, and advanced populations, were examined in order to elucidate the relationship between position and frequency distribution of chiasmata and quantitative traits, including yield heterosis in common beans. The hybrid and advanced population groups were determined to possess 83% and 54% increased chiasma frequency, respectively in contrast to inbred lines. The increase in chiasma frequency of these populations was further manifested in a high number of interstitial chiasmata. The regular and superior chromosome behaviour of the hybrids was found to be positively associated with quantitative measures on bean yield, harvest index and bean yield efficiency. The results were discussed from the point of view that: a) increased interstitial chiasmata may provide an effective mechanism for maintaining genetic diversity and heterosis in hybrid populations; and b) heterosis for chiasma frequency and quantitative traits may be due to dispersed genes on the chromosomes having combined intra-and interallelic interactions. The data provide evidence for the existence of positive associations between interstitially localized chiasmata with its recombination potential and regular chromosome behaviour to bean yield heterosis. The role of enhanced interstitial chiasmata to promote higher levels of genetic variation and heterozygous advantage is discussed.     

QTL analysis of canning quality and color retention in black beans (Phaseolus vulgaris L.)

Black bean (Phaseolus vulgaris L.) consumption is increasing in the USA. One of the major challenges faced by breeders is to develop superior black bean cultivars to meet the demands of the canning industry. Processors require beans that take up water quickly during pre-canning soak and beans that retain their black color after canning. To properly assess canning quality requires expensive and detailed measurements of the canned product, often not possible for bean breeders. The objective of this research was identify quantitative trait loci (QTL) in a black bean recombinant inbred line (RIL) population for canning quality traits related to water uptake, color retention, and anthocyanin concentration. The parental lines from which the population was developed, Black Magic and Shiny Crow, contrasted in water uptake and color retention. These cultivars also differed in seed coat luster, controlled by a single gene, Asp. A medium-density linkage map of 1,449 markers and a distance of 1,660 cM was developed from this RIL population. The map was aligned to the bean genome sequence V1.0 by using sequence information associated with the Diversity Arrays Technology markers. QTL analysis revealed that the region near the Asp gene on chromosome Pv 07 is the major determinant of water uptake, explaining up to 49 % of the phenotypic variation. A group of QTL for color retention-related traits was found at the upper region of Pv 11, explaining up to 30 % of the phenotypic variation. A smaller effect QTL clustered on Pv 5 co-localized with a QTL for canned bean anthocyanin concentration and explained less than 10 % of the phenotypic variation. These color-related QTL have marker-assisted selection potential for bean breeders interested in enhancing color retention and anthocyanin concentration of processed black beans.     

Genetic diversity, inter-gene pool introgression and nutritional quality of common beans (Phaseolus vulgaris L.) from Central Africa

The Great Lakes region of Central Africa is a major producer of common beans in Africa. The region is known for high population density and small average farm size. The common bean represents the most important legume crop of the region, grown on over a third of the cultivated land area, and the per capita consumption is among the highest in the world for the food crop. The objective of this study was to evaluate the genetic diversity in a collection of 365 genotypes from the Great Lakes region of Central Africa, including a large group of landraces from Rwanda as well as varieties from primary centers of diversity and from neighboring countries of Central Africa, such as the Democratic Republic of Congo and Uganda, using 30 fluorescently labeled microsatellite markers and automated allele detection. In addition, the landraces were evaluated for their seed iron and zinc concentration to determine if genetic diversity influenced nutritional quality. Principal coordinate and neighbor-joining analyses allowed the separation of the landraces into 132 Andean and 195 Mesoamerican (or Middle American) genotypes with 32 landraces and 6 varieties intermediate between the gene pools and representing inter-gene pool introgression in terms of seed characteristics and alleles. Genetic diversity and the number of alleles were high for the collection, reflecting the preference for a wide range of seed types in the region and no strong commercial class preference, although red, red mottled and brown seeded beans were common. Observed heterozygosity was also high and may be explained by the common practice of maintaining seed and plant mixtures, a coping strategy practiced by Central African farmers to reduce the effects of abiotic and biotic stresses. Finally, nutritional quality differed between the gene pools with respect to seed iron and zinc concentration, while genotypes from the intermediate group were notably high in both minerals. In conclusion, this study has shown that Central African varieties of common bean are a source of wide genetic diversity with variable nutritional quality that can be used in crop improvement programs for the region.