New microsatellite markers isolated from mungbean (Vigna radiata (L.) Wilczek)

In this study, we reported the isolation and analysis of new polymorphic microsatellites in mungbean (Vigna radiata (L.) Wilczek). Twelve out of 210 primer pairs screened in 30 mungbean accessions gave polymorphism. The polymorphic markers detected two to three alleles per locus with an average of 2.08. Observed heterozygosity varied from 0 to 0.133, while expected heterozygosity ranged from 0.095 to 0.498. Tests for Hardy-Weinberg equilibrium (HWE) and pairwise linkage disequilibrium of the polymorphic loci revealed that all loci except MB-SSR14 significantly departed from HWE and four pairwise combinations, viz. MB-SSR14 vs. MB-SSR42, MB-SSR42 vs. MB-SSR87, MB-SSR114 vs. MB-SSR121, and MB-SSR175 vs. MB-SSR231 significantly deviated from linkage disequilibrium. The markers are being used to study genetic diversity and genome mapping of mungbean.     

Isolation and characterization of seven tetranucleotide microsatellite loci in mungbean,Vigna radiata

A simple and rapid method for isolating tetranucleotide microsatellites in mungbean, Vigna radiata, based on the 5′‐anchored polymerase chain reaction technique, revealed 15 microsatellite sequences. We report on the characterization of seven polymorphic microsatellite loci in V. radiata. The number of alleles per locus ranged from 2 to 6, whereas the observed heterozygosity ranged from 0 to 0.561. Tetranucleotide markers are useful because they amplify fewer stutter bands thus making scoring easier. These markers will be useful for detecting genetic variation in mungbean varieties for germplasm management and development of the crop.     

Chromatographic and electrophoretic methods for analysis of superoxide dismutases

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature, are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.     

Isolation of microsatellite markers in mungbean,Vigna radiata

A simple and rapid method for isolating microsatellite loci in mungbean, Vigna radiata, based on the 5′‐anchored polymerase chain reaction technique revealed 23 microsatellite loci and six cryptically simple sequence repeats. We report on the characterization of seven polymorphic microsatellite loci in V. radiata. The number of alleles per locus ranged from 2 to 5 while the observed heterozygosity ranged from 0 to 0.9048. These markers should prove useful as tools for detecting genetic variation in mungbean varieties for germplasm management and crossbreeding purposes.     

Unexpected plasticity of the quaternary structure of iron-manganese superoxide dismutases

Protein 3D structure can be remarkably robust to the accumulation of mutations during evolution. On the other hand, sometimes a single amino acid substitution can be sufficient to generate dramatic and completely unpredictable structural consequences. In an attempt to rationally alter the preferences for the metal ion at the active site of a member of the Iron/Manganese superoxide dismutase family, two examples of the latter phenomenon were identified. Site directed mutants of SOD from Trichoderma reesei were generated and studied crystallographically together with the wild type enzyme. Despite being chosen for their potential impact on the redox potential of the metal, two of the mutations (D150G and G73A) in fact resulted in significant alterations to the protein quaternary structure. The D150G mutant presented alternative inter-subunit contacts leading to a loss of symmetry of the wild type tetramer, whereas the G73A mutation transformed the tetramer into an octamer despite not participating directly in any of the inter-subunit interfaces. We conclude that there is considerable intrinsic plasticity in the Fe/MnSOD fold that can be unpredictably affected by single amino acid substitutions. In much the same way as phenotypic defects at the organism level can reveal much about normal function, so too can such mutations teach us much about the subtleties of protein structure.     

Screening and identification of random amplified polymorphic DNA (RAPD) markers linked to mungbean yellow mosaic virus (MYMV) resistance in mungbean (Vigna radiata (L.) Wilczek)

Bulk segregant analysis (BSA) and random amplified polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible VBN (Gg) 2 × resistant KMG 189 to screen and identify the molecular marker linked to mungbean yellow mosaic virus (MYMV) resistant gene in mungbean. Two DNA bulks namely resistant bulks and susceptible bulks were setup by pooling equal amount of DNA from five randomly selected plants of each disease response. A total of 72 random sequence decamer oligonucleotide primers were used for RAPD analysis. Primer OPBB 05 (5′-GGGCCGAACA-3′) generated OPBB 05 260 fragment in resistant parent and their bulks but not in the susceptible parent and their bulks. Co segregation analysis was performed in resistant and susceptible F2 individuals, it confirmed that OPBB 05 260 marker was tightly linked to mungbean yellow mosaic virus resistant gene in mungbean.     

Purification and characterization of tubulin from mung bean (Vigna radiata)

Tubulin has been purified from mung bean seedling by Zn²⁺-induced polymerization. Both α- and β-subunits of mung bean tubulin are different from those of brain tubulin in electrophoretic mobility, colchicine binding and peptide map. Heterogeneity of mung bean tubulin has also been documented suggesting diversification of tubulin despite its conserved nature in general.     

Differences in responses to iron deficiency among various cultivars of mungbean (Vigna radiata (L.) Wilczek)

Ten mungbean cultivars were evaluated for their resistance to iron deficiency in view of chlorosis symptoms, plant growth and seed yield under field conditions on a calcareous soil in Thailand. The KPS2 cultivar was highly susceptible; the KPS1, PSU1 and Pag-asa 1 cultivars were somewhat susceptible; the VC1163B cultivar was moderately tolerant; the CN36, CN60, UT1 and CNM-I cultivars were tolerant; and the CNM8509B cultivar was very tolerant to iron deficiency. Foliar application of a solution of 5 g L^-1 ferrous sulphate was effective in correcting chlorosis that was induced by iron deficiency, and it enhanced both the growth and the yield of susceptible cultivars. Compared with the susceptible cultivar KPS2, the tolerant cultivar UT1 had a greater ability to lower the pH of the nutrient solution in response to iron deficiency. The root-associated Fe³⁺-reduction activity of UT1 that had been grown in -Fe medium was similar to that of the plants grown in +Fe medium when the acidification of the medium occurred. Acidification of the medium in response to iron deficiency might contribute to the efficient solubilization of iron from calcareous soils, and it related more closely to the resistance to iron deficiency than Fe³⁺ reduction by roots in mungbean cultivars.     

Purification and regulation of aspartate transcarbamylase from germinated mung bean (Vigna radiata) seedlings

Aspartate transcarbamylase (EC 2.1.3.2) was purified to homogeniety from germinated mung bean seedlings by treatment with carbamyl phosphate. The purified enzyme was a hexamer with a subunit molecular weight of 20,600. The enzyme exhibited multiple activity bands on Polyacrylamide gel electrophoresis, which could be altered by treatment with carbamyl phosphate or UMP indicating that the enzyme was probably undergoing reversible association or dissociation in the presence of these effectors. The carbamyl phosphate stabilized enzyme did not exhibit positive homotropic interactions with carbamyl phosphate and hysteresis. The enzyme which had not been exposed to carbamyl phosphate showed a decrease in specific activity with a change in the concentration of both carbamyl phosphate and protein. The carbamyl phosphate saturation and UMP inhibition patterns were complex with a maximum and a plateau region. The partially purified enzyme also exhibited hysteresis and the hysteretic response, a function of protein concentration, was abolished by preincubation with carbamyl phosphate and enhanced by preincubation with UMP. All these observations are compatible with a postulation that the enzyme activity may be regulated by slow reversible association-dissociation dependent on the interaction with allosteric ligands     

Distribution of Cu2+-diamine oxidase during ontogeny of seedlings of Vigna radiata cultivars

Activity of Cu2+-diamine oxidase (DAO; E.C.1.4.3.6.) was measured in Vigna radiata (L.) Wilczek cultivars K851, MH8320 and Pusa Baisakhi in light and in dark during ontogeny of seedlings. DAO activity was always the highest in cv. K851. In both light and dark grown seedlings maximum DAO activity was detected on day 2 after germination. Thereafter, in light grown seedlings it declined consistently upto non-detectable levels. In dark, DAO activity was higher than in light and it had the second maximum on day 7 following a similar declining pattern as observed for the light grown seedlings. The DAO activity was higher in a shoot apex alongwith leaves than in roots and shoot axis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and characterization of high salt-soluble vicilin from mung bean (Vigna radiata)

We report a method for the purification of vicilin from mung bean (Vigna radiata) mainly on the basis of solubility of mung bean vicilin even in high salt. Mung bean vicilin remains in solution even after 90% relative saturation of ammonium sulphate. The resulting supernatant after dialysis was subjected to gel filtration (Sephadex G-150) to remove other contaminant polypeptides, and finally the protein was purified by DEAE cellulose chromatography. This purified fraction exhibited 3 bands on SDS-PAGE compared with vicilin from other legumes which exhibite more than 3 bands generally. The results raise the possibility that the presence of the two small polypeptides in vicilin preparations is the breakdown product of the major larger one of mol.wt. 52 K and that vicilin may be a tetramer of four subunits of Mr 52000. That the high salt-soluble protein containing 52 K subunit is vicilin has been determined by several criteria.     

Chlorophyll mutations in mungbean (Vigna radiata (L.) Wilczek)

Summary Twenty mutants isolated from Latisail, Jhingasail and Pankaj varieties of rice (Oryza sativa L.) were screened for two aspects of nutritive quality, namely crude protein content and distribution pattern of protein in the endosperm. Observations revealed a wide variation for both characters, and while there was no consistent association between protein content and test grain weight, which varied between varieties, a positive correlation between protein content and grain sterility was noted. In a few mutants protein distribution was observed to be varied and showed a similarity to optimum milling characteristics.     

Plant regeneration from cotyledonary node explants of mungbean (Vigna radiata (L.) Wilczek)

Sexually-mature mungbean (Vigna radiata (L.) Wilczek) plants were efficiently regenerated from cotyledonary node explants. The explants were capable of directly developing multiple shoots on basal media devoid of any growth regulators. The shoot multiplication was influenced by media composition, growth regulators, age of donor seedling and explant type. The explants with both the cotyledons attached to the embryonic axis excised from 4-d-old seedlings, produced the highest number of shoots (5 or 6) in 100% of the cultures within 2 weeks on B₅ basal medium (BBM) containing BAP or 2-iP, respectively, (at 5x10^–7M) and 3% sucrose. Shoots elongated and developed better using BAP. Increasing micronutrients, carbohydrate and nitrogen levels in the medium above the original formulation of B₅ basal medium appeared to be of no benefit for increasing the number of shoots. The shoots were rooted on basal MS medium or MS containing 10^–6 of NAA, IAA or IBA. This protocol was found applicable to six other cultivars of mungbean. One hundred rooted shoots were successfully established in soil in the glasshouse, where 90% of them survived. The regenerated plants flowered precociously, but produced normal pods and viable seeds.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - 2-iP 6- — -dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium - B₅ Gamborg et al. (1968) medium - C medium MS salts + B₅ vitamins     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.     

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I.     

Purification and characterization of an allosteric fructose-1,6-bisphosphate aldolase from germinating mung beans (Vigna radiata)

Cytosolic fructose-1,6-P(2) (FBP) aldolase (ALD(c)) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 micromol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD(c))-IgG. The enzyme's native M(r) was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg(2+) had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65 degrees C. It demonstrated: (i) a temperature coefficient (Q(10)) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD(c) is bifunctional for FBP and sedoheptulose-1,7-P(2) (K(m) approximately 17 microM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K(i) values=2.2, 3.1 and 7.5mM, respectively). By contrast, the addition of 2mM ATP: (i) reduced V(max) by about 2-fold, (ii) increased K(m)(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h=1.0 and 2.6 in the absence and presence of 2mM ATP, respectively). Potent feedback inhibition of ALD(c) by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.