Nutritional and nutraceutical potential of rice bean (Vigna umbellata) -a legume with hidden potential.

In the recent years there has been paradigm shift in global agriculture for the exploration of different underutilized crops as future potential crops. Rice bean [Vigna umbellata (Thunb.) Ohwi and Ohashi] one of the lesser known pulses among Vigna species has gained attention during last decade as food and nutritional security crop. Rice bean seeds are well-balanced source of beneficial constituents such as protein, carbohydrates, minerals, vitamins, polyunsaturated fatty acids (PUFAs) and anti-oxidants for health benefits and combating malnourishment in human. In the present investigation, seeds of 15 diverse rice bean accessions from north-western Himalayan region were analyzed for nutrients, anti-nutrients and nutraceutical traits. Significant differences were observed among genotypes for different traits. The rice bean genotypes revealed variation for major quality traits including total carbohydrates (50.56-56.87%), crude protein content (22.56-25.97%) and lipid content (1.87 to 3.17%) with the higher proportion of linolenic acid followed by linoleic acid which are nutritionally desirable PUFAs. The genotype IC-548758 revealed higher proportion of desirable quality traits. Among protein fractions, globulins and albumins constituted major seed storage protein fraction in rice bean seeds. The wide range variation was also observed for anti-nutrients like including raffinose family oligosaccharides (RFOs), phenolics, tannins, trypsin inhibitor (TI), phytic acid, lipoxygenase activity and saponin content among genotypes. Insignificant correlation among iron, zinc, magnesium and manganese revealed good selection accuracy for genetic biofortification program in rice bean. In summary, the genotype IC-548757, IC-548760 and IC-548770 revealed lower proportion of anti-nutrients, whereas, the genotype IC-548759 and IC-548757 revealed higher level of free radical scavenging activity indicating nutritional and nutraceutical superiority of these genotypes. Overall, the study revealed nutritional superiority of genotype IC-548770, IC-548758 and IC-548760 with balanced proportions of nutrients and anti-nutrients. Rice bean legume has the potential to support more sustainable and resilient food and nutritional security in future. Our study highlights the potential of different rice bean genotypes as functional ingredients for future food and nutritional security programmes.

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding ß-D-Galactose, N-Acetyl-D-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata).

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843 bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5 mM IPTG after 4 h incubation at 37 °C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35 kDa m.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26 mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of ß-pleated sheets and ß-turns in RbL protein structure. ß-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Research advances and prospects of legume lectins.

Lectins are widely distributed proteins having ability of binding selectively and reversibly with carbohydrates moieties and glycoconjugates. Although lectins have been reported from different biological sources, the legume lectins are the best-characterized family of plant lectins. Legume lectins are a large family of homologous proteins with considerable similarity in amino acid sequence and their tertiary structures. Despite having strong sequence conservation, these lectins show remarkable variability in carbohydrate specificity and quaternary structures. The ability of legume lectins in recognizing glycans and glycoconjugates on cells and other intracellular structures make them a valuable research tool in glycomic research. Due to variability in binding with glycans, glycoconjugates and multiple biological functions, legume lectins are the subject of intense research for their diverse application in different fields such as glycobiology, biomedical research and crop improvement. The present review specially focuses on structural and functional characteristics of legume lectins along with their potential areas of application.

Evaluation and characterization of trypsin inhibitor from rice bean with inhibitory activity against gut proteases of Spodoptera litura.

Trypsin inhibitor (TI) in rice bean (Vigna umbellata) varied spatio-temporally in different parts of the plant, with the highest level (30.9 mg/g d.w.) noted in the maturing seeds of genotype BRS-2 at 160 days after planting (DAP). The TI from rice bean seeds was isolated and purified approximately 182-fold, with a final yield of 29% using ammonium sulfate precipitation, ion exchange chromatography through DEAE-Sepharose, gel permeation through Superdex-75, and finally by affinity chromatography using a trypsin-Sepharose column. The purified TI showed a single band on SDS-PAGE under reducing conditions with an apparent molecular mass of 24 kDa. The highest activity of purified inhibitor (about 90%) was recorded at pH 4.0 at 37 °C, suggesting the stability of the inhibitor under acidic conditions. The TI exhibited an inhibitory effect against Spodoptera litura larvae. A progressive decline in larval weight, growth, and survival rate of larval development was observed after feeding S. litura larvae on a diet supplemented with increasing concentrations of rice bean TI. The highest TI content in the seeds nearing maturity correlates to the role of TIs in protecting against insect pests. The study clarifies the role of rice bean protease inhibitors as a potential strategy against insectpests of economic importance.

Genetic and biochemical diversity among Valeriana jatamansi populations from Himachal Pradesh.

Valeriana jatamansi Jones is an important medicinal plant that grows wild in Himachal Pradesh, India. Molecular and biochemical diversity among 13 natural populations from Himachal Pradesh was assessed using RAPD and GC-MS to know the extent of existing variation. A total of seven genetically diverse groups have been identified based on RAPD analysis which corroborated well with the analysis based on chemical constituents. The essential oil yield ranged from 0.6% to 1.66% (v/w). A negative correlation between patchouli alcohol and viridiflorol, the two major valued constituents, limits the scope of their simultaneous improvement. However, other few populations like Chamba-II and Kandi-I were found promising for viridiflorol and patchouli alcohol, respectively. The analysis of chemical constitution of oil of the populations from a specific region revealed predominance of specific constituents indicating possibility of their collection/selection for specific end uses like phytomedicines. The prevalence of genetically diverse groups along with sufficient chemical diversity in a defined region clearly indicates the role of ecology in the maintenance of evolution of this species. Sufficient molecular and biochemical diversity detected among natural populations of this species will form basis for the future improvement.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.

RNAi for insect control: current perspective and future challenges.

The research on the RNA interference (RNAi) for the control of insect pests has made significant growth in recent years. The availability of the genomic sequences of insects has further widened the horizons for the testing of this technology to various insect groups. Different modes of application of double-stranded RNA (dsRNA) have been tested; however, the practicability of delivery of dsRNA in insects still remains the biggest challenge. Till date, the oral delivery of dsRNA in insects is one of the efficient approaches for the practical application of this technique. The uptake of dsRNA from the insect gut is mediated either by SID-1/SID-2 transmembrane proteins or by endocytosis; however, the systemic RNAi machinery still remains to be revealed in insect species. The RNAi-mediated gene knockdown has shown striking results in different insect groups, pointing it to be the upcoming technique for insect control. However, before the successful application of this technique for insect control, some potential issues need to be resolved. This review presents the account of prospects and challenges for the use of this technology for insect control.

Advances in RNA interference technology and its impact on nutritional improvement, disease and insect control in plants.

This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

Nutritional potential of rice bean (Vigna umbellata): an underutilized legume.

UNLABELLED: Rice bean, a less known and underutilized legume, has emerged as a potential legume because of its nutritional potential. The nutritional quality of rice bean is higher as compared to many other legumes of Vigna family. In the present study, 16 diverse rice bean genotypes were evaluated for major nutritional constituents viz; protein content, total lipids, dietary fiber, total carbohydrates, vitamins, minerals, protein fractions, amino acid, and fatty acid profile. The protein content to the extent of 25.57% was observed in the genotype BRS-2 with in vitro digestibility of 54.23%. The fatty acid profile revealed the higher percentage of unsaturated fatty viz., linoleic and linolenic acid, which are nutritionally desirable in the diet. Albumins (6.13% to 7.47%) and globulins (13.11% to 15.56%) constituted the major portion of proteins. Anti-nutritional factors were in the range of: total phenolics (1.63% to 1.82%), total tannins (1.37% to 1.55%), condensed tannins (0.75% to 0.80%), hydrolysable tannins (0.56% to 0.79%), trypsin inhibitor (24.55 to 37.23 mg/g), phytic acid (7.32 to 8.17 mg/g), lipoxygenase activity (703 to 950 units/mg), and saponin content (1.2 to 3.1 mg/100 g). The oligosaccharides associated with the production of flatulence viz., raffinose, stachyose, and verbascose were in the limits of 1.66% to 2.58%, 0.94% to 1.88%, and 0.85% to 1.23%, respectively. In vitro protein digestibility up to 55.57% was observed in rice bean genotypes. The present study has revealed that rice bean is a nutritionally rich legume as compared to many other legumes of the category. Among different genotypes BRS-2 was observed superior and could be advocated for consumption as well as for inclusion in crop improvement programs. PRACTICAL APPLICATION: Rice bean is nutritionally rich legume, but despite its nutritional excellence, it has been put in underutilized category. Because of this and several other reasons the people are not aware of its nutritional benefits. Moreover, the complete nutritional details are also not available on this pulse. The present study gives the vivid description of nutritional attributes of this legume for making people aware of its nutritional excellence and provoking improved work in rice bean.

RNA interference: a promising technique for the improvement of traditional crops.

RNA interference (RNAi) is a homology-dependent gene-silencing technology that involves double-stranded RNA directed against a target gene. This technique has emerged as powerful tool in understanding the functions of a number of genes in recent years. For the improvement in the nutritional status of the plants and reduction in the level of antinutrients, the conventional breeding methods were not completely successful in achieving the tissue-specific regulation of some genes. RNAi has shown successful results in a number of plant species for nutritional improvement, change in morphology and alteration in metabolite synthesis. This technology has been applied mostly in genetic engineering of important crop plants, and till date there are no reports of its application for the improvement of traditional/underutilized crops. In this study, we discuss current knowledge of RNAi function and concept and strategies for the improvement of traditional crops. Practical application. Although RNAi has been extensively used for the improvement of popular crops, no attention has been given for the use of this technology for the improvement of underutilized crops. This study describes the importance of use of this technology for the improvement of underutilized crops.