Vigna Radiata Seeds Research Articles

Role of apoplastic calcium during germination and initial stages of seedling establishment in Vigna radiata seeds

Calcium (Ca2+) is implicated in the initial phase of seed germination and seedling establishment. It is stored complexed with phytic acid during seed development and released by phytase action during germination. We observed phytase activity 18 h post-imbibition (PI) in Vigna seeds, while radicle protrusion occurred approximately 12 h PI. Cotyledon protein extracts prepared 4, 8, 16 and 24 h PI, subjected to Ca2+ immobilized metal affinity chromatography (Ca2+ IMAC), revealed the presence of Ca2+ binding proteins (CaBPs), while Ca2+-dependent amylase activity peaked 18 h PI, implying Ca2+ presence before its release from Ca-phytate, indicating an alternative source of Ca2+. Vigna cotyledon cell-wall preparations 4 h and 24 h PI, titrated against alkali, revealed high cation-binding capacity, and seeds 4 h PI demonstrated high rates of H+ extrusion. Ca2+-binding capacity as well as cell-wall bound Ca2+, measured in cotyledon cell-wall preparations from unimbibed seeds as well as seeds 24 h PI, using a novel competitive chelation technique, showed a marked decline in Ca2+ binding capacity, as well as cell-wall bound Ca2+. Imbibition in the presence of chelators, Ca2+-channel blockers, and H+-pump inhibitors, interfered with germination and radical extension. Further, EDTA-treated cotyledon protein extracts separated on Ca2+IMAC showed a larger CaBP peak than control cotyledon extracts. Pooled fractions clearly showed Ca2+-induced extrinsic fluorescence with anilino -napthalene sulfonate. The results strongly implicate the apoplast may be a major source of Ca2+ in the initial phase of germination and seedling establishment in Vigna seeds.

NiO nanodisks: Highly efficient visible-light driven photocatalyst, potential scaffold for seed germination of Vigna Radiata and antibacterial properties

Abstract In the present study, we report the facile synthesis, detailed characterizations, photocatalytic, seed germination and antibacterial applications of NiO nanodisks. The nanodisks were fabricated by simple hydrothermal method and further characterized in detail which shows the well-crystalline cubic phase of NiO nanodisks with pure and large density growth. The crystal quality and purity of the NiO nanodisks were examined by calcining them at different temperatures (i.e 300 °C, 400 °C and 500 °C). NiO nanodisks with average size of 5.1 nm were obtained at 300 °C calcination, whereas, 6.8 nm and 12.7 nm sized NiO particles were obtained at 400 °C and 500 °C respectively. The synthesized nanodisks were used as proficient visible-light driven photocatalyst for the photocatalytic degradation of various harmful dyes. The detailed photocatalytic experiments revealed that under visible light irradiation methylene blue dye degraded up to 98.7% in just 20 min whereas the mixture of other dyes were degraded up to 94–97.6%. The prepared nanodisks have also demonstrated extremely high stability and recyclability (up to 4th cycle) for removing water contamination caused by different dyes and their mixtures. Additional, the NiO nanodisks were used as potential framework for the germination of Vigna Radiata seeds which exhibited very high (1500 ppm) antiproliferative activities with percentage inhibition growth values ranging from 12.6 to 46%. Finally, the antibacterial activities of NiO nanodisks were tested against Staphylococcus aureus and Escherichia coli.

Biosorption Capacity of Cr (VI) on Live and Dead Scenedesmus rubescens: Kinetic, Equilibrium and Phytotoxicity Study

The bisorption potential and kinetics of Cr (VI) using live and dead cells of Scenedesmus rubescens have been explored for the first time in the present work. Ideal conditions for the biosorption were found to be: pH 2 agitated at 90 rpm and at a dose of 0.8 g L−1 for both live and dead S. rubescens. For the initial Cr (VI) concentration ranging from 5 mg L−1 to 90 mg L−1, the experimental results showed that the metal adsorption capacity increased while the removal efficiency of Cr (VI) decreased with an increase in the initial Cr (VI) concentration. The results fitted well with Sips and Redlich-Peterson isotherm models and the biosorption mechanism was explained well with the pseudo-second-order model. Lastly, the phytotoxicity assay of the treated aqueous solution was performed on the Vigna radiata seeds, which confirmed its suitability for agricultural purpose.

Use of a germination bioassay to test compost maturity in Tekelan Village

Livestock waste from cattle farms in Tekelan village, Getasan Subdistrict, Semarang Regency can be grouped into three types, namely solid waste, slurry and waste water. Solid waste (cow dung) was processed into compost, while slurry and waste water were used to make liquid fertilizer. This compost was used as a component of planting media in horticultural crops and potted plants production. We evaluated the toxicity (phytochemical and ecotoxicological) test of compost by using germination index (GI). Vigna radiata seeds are sown on filter paper dampened with compost extract for different times. GI was calculated by relative germination (G) and relative radical length (L). The germination index (GI) = G / G0 x L / L0 x 100, where G0 and L0 are values obtained by distilled water as a control. The results showed that germination bioassay and radical length using aquades and groundwater in Tekelan village did not affect the radical length of Vigna radiata . Technically, groundwater in Tekelan village can be used as a germination bioassay control. The cow dung compost substrate appears to have a major influence on compost toxicity. Mature compost was produced on day 14 with a GI of 104.03.

Inheritance and a major quantitative trait locus of seed starch content in mungbean (Vigna radiata (L.) Wilczek)

Mungbean (Vigna radiata) seeds are used for direct consumption by people and are raw material for sprout, starch and noodle industries in Asia. High-yielding mungbean cultivars with high seed starch content are preferred for the starch and noodle industries. At present, there is no report on genetic control of seed starch content in mungbean. The objective of this study were to (i) estimate heritability of the starch content in mungbean and (ii) locate a major quantitative trait locus (QTL) controlling starch content. An F2 population of 123 individuals was developed from the cross V6087AG (high seed starch) × V5020BY (low seed starch). Seeds of F2 and F2:3 populations were determined for seed starch content. Broad-sense heritability estimated for seed starch content in the F2 and F2:3 populations were higher than 80%. Seed starch content showed a relatively high and significant correlation (r = 0.6) with seed weigh in both F2 and F2:3 populations. Bulk segregant analysis using 123 polymorphic SSR markers revealed that only SSR marker CEDG092 on mungbean chromosome 8 associated with the starch content. QTL mapping using CEDG092 and 22 newly developed SSR markers the chromosome confirmed that a major QTL, qSSC8.1, flanked by markers Vr08-SSR113 and Vr08-SSR114 control seed starch content in both F2 and F2:3 populations. qSSC8.1 showed no localization with seed weight QTL and explained about 12.34–13.84% of total variation of the seed starch content in the two populations. Allele(s) from V6087AG at this QTL increased seed starch content. Vr08-SSR113 and Vr08-SSR114 spanned a genome region of about 385 Kbp and there were 21 annotated genes in this region. This genome region can be used as target for fine mapping to identify gene controlling seed starch content in mungbean.

Electrochemical decolorization of methyl red by RuO2-IrO2-TiO2 electrode and biodegradation with Pseudomonas stutzeri MN1 and Acinetobacter baumannii MN3: An integrated approach

Textile effluent consists of enormous quantities of toxic dyes, which are being discharged into natural aqueous system and thus contaminate the water quality. Hence it is important to develop an eco-friendly and cost effective technology to treat the dyes contaminated wastewater. In this research, an integrated approach of electrochemical oxidation (EO) and biodegradation process (BP) was studied of methyl red (MR) dye. In EO, RuO2-IrO2-TiO2 is used as anode and titanium mesh electrode as cathode. This was followed by BP of the treated EO effluent. Various parameters viz., pH (5–10), sodium chloride concentrations (NaCl) (1–5 g L−1) and current density (10–30 mA cm2) were optimized. The results of the EO showed 99.96% of MR decolorization within 10 min at pH of 5, NaCl of 2 g L−1 and current density of 30 mA cm2. The EO treated MR was further treated by BP Pseudomonas stutzeri MN1, Acinetobacter baumannii MN3 and mixed consortia of MN1 and MN3. The out of three treatments, the results of mixed consortium BP showed 90% removal of COD at the end of 24 h. The phytotoxic evaluation using Vigna radiata seeds confirmed the toxicity of untreated MR solution, whereas, 100% germination was observed in treated (biodegraded) MR solution. Overall these results evidenced that MR dye was completely decolorized and mineralized by EO and BP within 10 min and 24 h respectively. Hence, this integrated approach can be used as an effective degradation method to treat dyes in the textile industry.

Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds

Dipeptidyl peptidases (DPPs) are widely distributed exopeptidases that hydrolyse the dipeptide moieties from the N-termini of oligopeptide chains. In the present study, DPP-I was purified from germinated moong bean seeds via acid and ammonium sulphate precipitation followed by successive chromatographies, that is, gel filtration (pH 7.4), cation exchange (pH 5.9) and anion exchange (pH 7.5). The purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and in situ gel assay. Purified plant DPP-I is a monomeric enzyme with a molecular weight of 38kDa. It works optimally at pH 7.0 and 40°C, and it exhibits stability at pH ranging from acidic to slightly alkaline. Plant DPP-I preferentially hydrolyses glycine–arginine–4-methoxy-β-naphthylamide and various other synthetic dipeptidyl substrates, but none of the studied endopeptidase and monopeptidase substrates. Inhibitory studies revealed the role of Cys and His amino acids in the catalytic mechanism. Functional studies of DPP-I revealed the significant role of this glycoproteinous enzyme in protein mobilization during germination.

Sodium fluoride (NaF) induced changes in growth and DNA profile of Vigna radiata

Fluoride (F) is a natural, ubiquitous, non-biodegradable and hazardous pollutant. To investigate its effect, Vigna radiata seeds were treated with NaF (0, 25, 50, 75, 100 mg NaF kg-1) and its effect was studied on germination, growth and RAPD profile. The variation in DNA profile in response to NaF treatment was detected by RAPD-PCR technique. The result of RAPD was observed in terms of GTS% (Genomic Template Stability). Results showed that there was gradual decrease in GTS% i.e. increase in DNA damage from 25 to 75 mg NaF kg-1 concentration. These data demonstrate that RAPD is a reliable tool and permits greater insights into the genetic alteration of V. radiata .

Seed germination test as an alternative urine-based non-invasive pregnancy test in alpacas (Vicugna pacos)

The goal of this study was to assess the seed germination test as a pregnancy diagnostic method in alpacas (Vicugna pacos). Sampling was carried out in 6–8 week intervals from April 2013 to February 2014 on three private farms in the Czech Republic (Central Europe). The urine was collected non-invasively by catching it in plastic cups during spontaneous urination. In total, five urine samples were obtained from each of the 12 tested alpacas. Two urine concentrations were tested, and the urine was diluted by distilled water in ratios of 1: 4 and 1: 14. Fifteen millilitres of the urine-water solution was applied onto 50 mung bean (Vigna radiata) seeds in Petri dishes. Germination rates were counted two days after establishment of the experiments. Lengths of the shoots were measured on the fifth day. It was determined that alpaca urine significantly inhibited germination and growth of seeds in general. The inhibitory effect was higher with higher concentrations of urine. However, seeds germinated and grew better in the urine of pregnant females than in urine of non-pregnant females. While further research is needed, the seed germination test with mung beans seems to be a viable method for pregnancy diagnosis in alpacas.

Purification and biochemical characterization of dipeptidyl peptidase-II (DPP7) homologue from germinated Vigna radiata seeds

Dipeptidyl peptidases (DPPs) are potent exopeptidases, which possess central role in proteolysis. As compared to other members of DPP family, proline containing dipeptide hydrolysing activity of DPP-II (Dipeptidyl peptidase II) is unique as it hydrolyses imino group and plays a key role in protein metabolism. In present study, DPP-II was purified from germinated moong bean seeds using acid and ammonium sulphate precipitation followed by successive chromatographies on gel filtration (pH 7.4) and cation exchanger (pH 5.9). Native PAGE and in-situ gel assay confirmed the apparent homogeneity. Purified plant DPP-II is an oligomeric enzyme with molecular weight of 97.3kDa. Highest DPP-II activity was observed at pH 7.5 and 37°C, with stability in the range of neutral to alkaline pH. Substrate specificity showed consequent activity for proline containing dipeptide followed by Lys-Ala and other hydrophobic dipeptides, but none of the studied endopeptidase and monopeptidase substrate was hydrolysed. Catalytic characterization with modifier studies revealed the involvement of Ser and His residues in its catalytic mechanism. Its dipeptidyl peptidase activity for proline containing dipeptide supported its role in the bioactive peptide generation and food industry. Functional studies of DPP-II revealed the significant involvement of this glycoproteinous enzyme in protein mobilization during germination. Further studies on industrial applications exploring physiological role are in progress.

Role of peroxidase activity and Ca(2+) in axis growth during seed germination.

Axis growth during seed germination is mediated by reactive oxygen species and apoplastic peroxidase plays a role by producing OH(·) from H2O2. Ca (2+) activates both apoplastic peroxidase and NADPH oxidase. Role of reactive oxygen species (ROS) in seed germination and axis growth has been demonstrated in our earlier works with Vigna radiata seeds by studying superoxide generation and its metabolism in axes (Singh et al. in Plant Signal Behav doi: 10.4161/psb.29278 , 2014). In the present study, the participation of apoplastic peroxidase along with the involvement of Ca(2+) in axis growth during germination and post-germination stage has been investigated. Pharmacological studies using peroxidase (POX) inhibitors (salicylhydroxamic acid, SHAM; sodium azide, NaN3) and OH(·) scavenger (sodium benzoate, NaBz) indicated that seed germination and early axis growth (phase I) depend much on POX activity. Subapical region of axes corresponding to radicle that elongated much particularly in phase II suggested high POX activity as well as high NADPH oxidase (Respiratory burst oxidase homologue, Rboh, in plants) activity as indicated from localization by staining with TMB (3,3',5,5'-tetramethyl benzidine dihydrochloride hydrate) and NBT (nitroblue tetrazolium chloride), respectively. Apoplastic class III peroxidase (Prx) and also cellular POX activity reached maximum at the time of radicle emergence as revealed by TMB staining, spectrophotometric and in-gel assay for POX activity. Treatment with Ca(2+) antagonists (La(3+), plasma membrane-located Ca(2+) channel blocker and EGTA, Ca(2+) chelator in apoplast) retarded seed germination and strongly inhibited axis growth, while Li(+) (blocks endosomal Ca(2+) release) was effective only in retarding phase II axis growth suggesting an involvement of Ca(2+) influx during early axis growth. From the effect of Ca(2+) antagonists on the localization of activities of POX and Rboh using stains, it appears that Ca(2+) plays a dual role by activating Prx activity in apoplast while activating Rboh by entering into cytosol.

Involvement of melatonin applied to Vigna radiata L. seeds in plant response to chilling stress

Abstract The impact of melatonin (50 µM L−1) applied to Vigna radiata seeds by hydro-priming on phenolic content, L-phenylalanine ammonialyase (PAL) activity, MEL level, antioxidant properties of ethanol extracts as well as electrolyte leakage from chilled and re-warmed Vigna radiata roots of seedlings were examined. Seedlings obtained from non-primed seeds, hydro-primed and hydro-primed with MEL were investigated after 2 days of chilling and 2 days of re-warming. At 25°C, the level of MEL in roots derived from seeds hydro-primed with MEL was 7-fold higher than in roots derived from non-primed seeds. However, the content of MEL significantly decreased in all variants investigated after re-warming, in contrast to PAL activity and phenolic levels, which reached the highest values. The antioxidant capacity of ethanol extracts from chilled and re-warmed roots, determined by ABTS+· assay, was correlated with phenolic content while the reducing ability of these extracts, determined by the FRAP method, correlated with PAL activity. However, both were the highest in rewarmed roots with applied MEL, which was accompanied by a significant decline in electrolyte leakage. Taken together, results may indicate that MEL can play a positive role in plant acclimation to stressful conditions and activation of phenolic pathway by this molecule.

Isolation, purification and biochemical characterization of dipeptidyl peptidase-III from germinated Vigna radiata seeds

Genome analysis of plants indicated majority of putative protease genes that need characterization at enzymatic and molecular level. Proteases execute important role in seed development but knowledge of dipeptidylpeptidases in seeds is limited. A dipeptidylpeptidase-III that cleaves Arg-Arg from Arg-Arg-4mβNA was purified from germinated Mung bean seeds. Screening of cereals and legumes for DPP-III revealed highest activity in Phaseolus vulgaris and Vigna radiata (variety P.9531) seeds germinated for 48h. Homogenate (10%) was acid precipitated upto pH 4.2 and then subjected to 0–55% ammonium sulphate precipitation followed by successive chromatographies on cation exchanger, gel filtration and anion exchanger. Enzyme purity was confirmed by native polyacrylamide gel electrophoresis and in situ gel assays. DPP-III is a heterodimer of ∼60kDa with two subunits of 32.8kDa and 27kDa on SDS-PAGE. DPP-III worked optimally at pH 8.0 and at 37°C with pH stability between 7.5 to 9.5. Arg-Arg-4mβNA was preferred substrate and none of endopeptidase and monopeptidase substrates were hydrolysed. Inhibitors studies revealed it as thiol protease with involvement of metals at active site. The enzyme might be involved in plant's development by generating/deactivating bioactive peptides. Further studies on functional characterization and molecular structure are in progress in our laboratory.

English

In the present study mono and mixed culture bacterial combinations of Azotobacter were used to inculcate Vigna radiata seeds. In general, bacterial inoculations (mono and mixed cultures) promoted seed germination, early growth parameters, auxin content, soluble protein content, peroxidase and acid phosphatase activity relative to non-inoculated control seedlings. Increase of (23%) as well as decrease (6.8%) in the root length were observed with bacterial inoculations relative to non-inoculated seedlings. About 20.07% enhancements in fresh weight and 62% enhancement in dry weight was observed in case of bacterial inoculations. Among the monoculture Ab-4(A3) induced pronounced growth stimulator effects, mixed culture combinations B6, B7, and B10 showed pronounced synergistic effects relative to respective monocultures, while B2 exhibited negative in majority of the parameters being studied.   Key words: Azotobacter, Vigna radiata, auxin, acid phosphatase, peroxidase. .

A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization

Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.

Non-enzymatic protein modification by the Maillard reaction reduces the activities of scavenging enzymes in Vigna radiata.

The non-enzymatic modification of proteins through the Maillard reaction plays an important role in the loss of seed viability during seed storage. In the present study we examined whether the Maillard reaction reduces the activities of scavenging enzymes in Vigna radiata (mung bean) seeds during storage. Seeds were stored under various conditions for different duration. Maillard products were monitored by measuring protein fluorescence, and the activities of glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POX) were determined. The accumulation of Maillard products in seed axes increased during storage with increasing moisture content and temperature, and was correlated with the decline in seed vigour. The activities of GR, CAT and APX decreased in proportion to the increase in Maillard products at all the moisture contents and temperatures tested. These enzymatic changes were also correlated with seed vigour. However, the activities of SOD and POX remained unchanged and appeared to be less sensitive to the Maillard reaction.

Effect of methionine on longevity, antioxygenic enzymes and lipid peroxides in Callosobruchus maculatus F.

The effect of methionine on longevity, antioxygenic enzymes and lipid peroxides in Callosobruchus maculatus was investigated using C. maculatus maintained on Vigna radiata seeds soaked in seven levels of methionine, 0, 1, 3, 5, 7, 9 and 12 mg/ml water. The life span and activities of catalase and peroxidase were higher in Callosobruchus maculatus infesting Vigna radiata seeds soaked in methionine as compared to their control cohorts. However, the lipid peroxide (malondialdehyde; MDA) levels generally remained unaltered. The present observation on the increased longevity in C. maculatus with methionine level is an extension of earlier findings on its anti-ageing effect in higher animals.

Influence of pre‐emergence experience on response to host and host plant odours in the larval parasitoid Eupelmus vuilleti

AbstractThe response to different host and plant species odours was investigated in Eupelmus vuilleti (Crw). This hymenopteran is a solitary ectoparasitoid of several species of bruchids developing inside Leguminosae seeds. The locomotor behaviour of females reared on Bruchidius atrolineatus (Pic) larvae developing in Vigna unguiculata (Walp) seeds was analysed using a tubular olfactometer. Females showed a specific sensitivity to the semiochemicals emanating from the host and the seed species on which they had developed. Odours from V. unguiculata seeds were attractive to the parasitoid and stimulated their locomotor activity. Odours from Vigna radiata (Wil) seeds had no effect on the locomotor behaviour. Odours from B. atrolineatus larvae were attractive to the females whereas odours from Callosobruchus maculatus (Fab), another bruchid species, had no effect. By isolating the females from the seed and the host at different developmental stages, we found that the specific sensitivity observed resulted from an early adult learning. This learning which occurs before the emergence from the seed while the imago is in the larval chamber of its host is dependent on contact with the seed and the host larval remains.

γ-Glutamyl peptides of Vigna radiata seeds

It has been reported elsewhere that Vigna radiata seeds contain a high concentration of γ-glutamyl- S-methylcysteine and its sulphoxide and that these compounds serve as index compounds for the chemotaxonomy of V. radiata. Beside these two γ-glutamyl peptides, nine other γ-glutamyl derivatives, of which three are new compounds, have been isolated and identified from V. radiata seeds. The structures of the new peptides, γ-glutamyl- S-methylcysteinyl-β-alanine ( S-methylhomoglutathione), γ-glutamyl- N δ-acetylornithine and γ-glutamyl-γ-glutamyl- S-methylcysteine, were confirmed by direct comparison with synthetic specimens. The other six γ-glutamyl derivatives were homoglutathione, γ-glutamyl glutamic acid, -aspartic acid, -phenylalanine, -leucine and -isoleucine.