Effects of winged bean tuber (WBT) modified starches substituted for cassava chip in concentrate diets on rumen fermentation, nutrient utilization, and blood metabolites in Thai native beef cattle.

Objective: This study examined the effects of substituting winged bean tuber steam (WBTS) modified starches for cassava chips (CSC) in the concentrate diet on rumen fermentation, nutrient utilization, and blood metabolites in Thai-native beef cattle. Methods: Four Thai-native bulls were assigned randomly as a 4 × 4 Latin square design, which represents the amount of CSC replaced with WBTS in the concentrate mixture diets at 0, 10, 20, and 30%. Results: Increasing levels of WBTS replacement for CSC in the concentrate diets had a quadratic effect on total dry matter (DM) intake (p<0.05). Replacement of WBTS at 20% and 30% for CSC did not alter total DM intake compared to 0% WBTS, whereas 10% WBTS replacement could significantly increase total DM intake by 0.41 kg DM/day compared to the control group. In addition, neutral detergent fiber (NDF) digestibility showed a quadratic increase (p < 0.05) when CSC was substituted at various levels of WBTS in the concentrate diet (p < 0.05). Replacement of CSC with WBTS at 10% and 20% showed higher NDF digestibility when compared to 0% replacement. There was a quadratic increase in blood glucose at 4 h post-feeding, and the average blood glucose value was significantly lower (p < 0.01) when substituting CSC with WBTS. Substituting WBTS for CSC at 10% in the concentrate diet showed the highest blood glucose concentration when compared to other treatments. Replacing CSC with WBTS at 10% and 20% shows a higher concentration of C3 than those of other treatments (0% or 30%). The nitrogen (N) intake (NI) increased linearly (p<0.05) when substituting WBTS for CSC at all levels in the diet. Additionally, N retention (NR) and the ratio of N retention to N intake increased (p<0.05) when substituting WBTS for CSC at 10%, 20%, and 30% compared to 0%. The gross energy intake (GEI), digestible energy intake (DEI), and energy efficiency (DEI/GEI) were quadratically increased when substituted with various levels of WBTS for CSC in the concentrate diet. Conclusion: The findings indicate that substituting 10% of CSC in the concentrate diet with WBTS may be sufficient as an alternative feed resource for improving nutrient utilization and metabolic efficiency in beef cattle diets.

First Report of False Rust caused by Synchytrium psophocarpi on Winged Bean (Psophocarpus tetragonolobus) in Taiwan.

From September 2020 to January 2021, an unknown disease of winged bean (Psophocarpus tetragonolobus) was reported by local growers in the Toucheng Town, Yilan County (N24.91, E121.85). The disease occurs in all age of winged bean, and the occurrence tended to be higher in humid environment, such as branches in lower canopy or branches in high density. The disease symptoms, which also appeared to be the sign of the pathogen, were spherical pustules in yellow to orange color on the stems, leaves, and pods of winged bean. Severely infected plants also exhibited growth reduction, malformation, and curling of the leaves and pods. According to the disease literature of winged bean, this unknown disease was likely to be the false rust caused by a chytrid pathogen, Synchytrium psophocarpi (UK, CAB International. 1993); and the uredinia-liked pustules could be the sori, which contain numerous ovoid to globose sporangia inside. In order to characterize the pathogen identity, the sori were manually ruptured to assess the size of individual sporangium, which had an average of 26.71 ± 4.25 µm x 26.61 ± 4.60 µm (n=42), similar to the size reported in literature (Drinkall and Price. 1979). To confirm the molecular identity, the full genomic sequences from the small subunit (SSU) to the internal transcribed spacer-1 (ITS-1), 5.8S unit, and ITS-2 were amplified using the primer sets NS3 and ITS4. The 2,263 bp amplicon was cloned and sequenced to reveal the identity (Smith et al. 2014). The BLASTN results matched the SSU of our isolate (MW649126.1) to the Synchytrium minutum (HQ324138.1) with 96% similarity (1,075 out of 1,121 bp in length), Synchytrium decipiens isolate DAOM_87618 (KF160868.1) with 92% similarity (1,215 out of 1,326 bp in length) and S. decipiens isolate AFTOL-ID 634 (DQ536475.1) with 92% similarity (1210 out of 1316 bp in length). Phylogenetic analysis using the SSU sequence revealed this unknown pathogen was the grouped within the clade of Synchytrium genus with 100% bootstrapping confidence (Smith et al. 2014). Accordingly, the pathogen was confirmed to be a Synchytrium chytrid fungus. To complete the Koch's postulates, the sori were collected from infected tissue. After vortexing washing in 1% bleach for surface sterilization, the sori were gently crashed by a plastic tube pestle to harvest sporangia. The sporangia were sprayed onto healthy winged beans cultivated in pots, and the inoculated plants were kept in a moisture bag in 25 °C. While leaf curling and malformation could be observed about 14 days post inoculation, the yellow to orange sori could be observed around 30 to 40 days post inoculation on the whole plants cultivated in pots. The sori were collected to confirm the sporangia and the sequences were identical to the original pathogen. Collectively, this study not only presents the first report for the false rust of winged bean in Taiwan, but also documents the first reference sequence of S. psophocarpi that will be useful for future molecular diagnosis. Since S. psophocarpi has been only reported in tropic regions including Indonesia, Malay Peninsula, Malaysia, Papua New Guinea, and Philippines, this report provides the first observation of S. psophocarpi moving in the subtropic region.

Comparison of Cassava Chips and Winged Bean Tubers with Various Starch Modifications on Chemical Composition, the Kinetics of Gas, Ruminal Degradation, and Ruminal Fermentation Characteristics Using an In Situ Nylon Bag and an In Vitro Gas Production Technique.

This research assessed the impact of cassava chips (CSC) and winged bean tubers (WBT) with various starch modification methods on the chemical composition, ruminal degradation, gas production, in vitro degradability, and ruminal fermentation of feed using an in situ and in vitro gas production technique. Experimental treatments were arranged for a 2 × 5 factorial, a completely randomized design with two sources of starch and five levels of modification treatments. Two sources of starch were CSC and WBT, while five modification treatments of starch were: no modification treatment, steam treatment, sodium hydroxide (NaOH) treatment, calcium hydroxide (CaOH2) treatment, and lactic acid (LA) treatment. The starch modification methods with NaOH and CaOH2 increased the ash content (p <0.05), whereas the crude protein (CP) content was lower after treatment with NaOH (p < 0.05). Steam reduced the soluble fraction (a) and effective dry matter degradability of WBT in situ (p <0.05). In addition, the WBT steaming methods result in a lower degradation rate constant in situ (p <0.05). The degradation rate constants for the insoluble fraction (c) in the untreated CSC were higher than those of the other groups. Starch modification with LA reduced in vitro dry matter degradability at 12 and 24 h of incubation (p <0.05). The starch modification method of the raw material showed the lowest pH value at 4 h (p <0.05). The source of starch and starch modification methods did not influence the in vitro ammonia nitrogen concentrations, or in vitro volatile fatty acids. In conclusion, compared to the CSC group and untreated treatment, treating WBT with steam might be a more effective strategy for enhancing feed efficiency by decreasing or retarding ruminal starch degradability and maintaining ruminal pH.

In Vitro Evaluation of Winged Bean (Psophocarpus tetragonolobus) Tubers as an Alternative Feed for Ruminants.

The purpose of the current study is to determine the effects of the replacement of cassava chips with winged bean (Psophocarpus tetragonolobus) tubers (WBTs) on gas production parameters, in vitro degradability, and ruminal fermentation in ruminant diets. The study was performed using a 3 × 4 factorial arrangements and was designed using a completely random method. Factor A employed three various roughage sources that were frequently used by locals to feed ruminants: Oryza sativa L. (a1), Brachiaria ruziziensis (a2), and Pennisetum purpureum (a3). The levels of WBTs substitutions for cassava chips at 0%, 33%, 66%, and 100% in the diet were selected as factor B. The experiment's findings revealed that replacing the cassava chips in the diet with WBTs at levels of 66 and 100% enhanced the fermentation process by producing a high gas volume at 96 h when Ruzi grass (RZ) was used as the main source of roughage (p < 0.01). The interaction between the roughage source and WBTs showed that organic matter (OM) degradability improved markedly in the case of RZ grass in combination with WBTs at all levels. Both the total volatile fatty acids (TVFAs) at 8 h of incubation and the average value decreased when a complete substitution of casava chips with WBT (WBT 100%) was employed or when employing rice straw as the main source of roughage (p < 0.01). There was no interaction between WBTs and roughage source on the ammonia-nitrogen (NH3-N) concentration (ml/dL) and rumen microbial count (p > 0.05). In summary, WBTs can be used effectively when combined with grass (Ruzi and Napier). The implementation of WBTs as a novel alternative feed may effectively replace cassava chips without affecting rumen function.

Reducing the biosynthesis of condensed tannin in winged bean (Psophocarpus tetragonolobus (L.) DC.) by virus-induced gene silencing of anthocyanidin synthase (ANS) gene.

The Underutilized legume-winged bean (Psophocarpus tetragonolobus (L.) DC.) and its various parts are infested with condensed tannin (CT) or proanthocyanidin (PA). CT has anti-nutritional effect as it adversely affects the digestion of proteins, minerals and vitamin among ruminants and humans. It is also responsible for low protein digestibility and decreased amino acid availability. One of the probable reasons of underutilization of P. tetragonolobus is due to its infestation with CT. Histochemical staining of various tissues of P. tetragonolobus with dimethylcinnmaldehyde (DMACA) developed a deep-blue colour indicating the presence of polyphenolic condensed tannin. Structural monomeric unit catechin and epi-catechin were reported to be responsible for biosynthesis of CT in P. tetragonolobus. The enzyme anthocyanidin synthase (ANS) and its corresponding transcripts were identified and phylogenetically mapped. The transcript was subjected to virus-induced gene silencing (VIGS) through agro-infiltration in P. tetragonolobus for reducing the CT-content. The WbANS-VIGS induced P. tetragonolobus resulted in four-fold decrease of CT as compared to the control P. tetragonolobus. A decrease of 73% of CT level was reported in VIGS silenced Wb-ANS line of P. tetragonolobus. This study resulted and confirmed that, the silencing of (ANS) gene in P. tetragonolobus has a regulatory effect on the condensed tannin biosynthesis. This study will pave way for further manipulation of ANS enzyme for reducing the biosynthesis of the anti-nutrient CT. Reducing the CT content will make this underutilized legume more acceptable. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03435-5.

The First Genetic Linkage Map of Winged Bean [Psophocarpus tetragonolobus (L.) DC.] and QTL Mapping for Flower-, Pod-, and Seed-Related Traits.

Winged bean [Psophocarpus tetragonolobus (L.) DC.] (2n = 2× = 18) is a tropical legume crop with multipurpose usages. Recently, the winged bean has regained attention from scientists as a food protein source. Currently, there is no breeding program for winged bean cultivars. All winged bean cultivars are landraces or selections from landraces. Molecular markers and genetic linkage maps are pre-requisites for molecular plant breeding. The aim of this study was to develop a high-density linkage map and identify quantitative trait loci (QTLs) for pod and seed-related traits of the winged bean. An F2 population of 86 plants was developed from a cross between winged bean accessions W054 and TPT9 showing contrasting pod length, and pod, flower and seed colors. A genetic linkage map of 1384 single nucleotide polymorphism (SNP) markers generated from restriction site-associated DNA sequencing was constructed. The map resolved nine haploid chromosomes of the winged bean and spanned the cumulative length of 4552.8 cM with the number of SNPs per linkage ranging from 36 to 218 with an average of 153.78. QTL analysis in the F2 population revealed 31 QTLs controlling pod length, pod color, pod anthocyanin content, flower color, and seed color. The number of QTLs per trait varied between 1 (seed length) to 7 (banner color). Interestingly, the major QTLs for pod color, anthocyanin content, and calyx color, and for seed color and flower wing color were located at the same position. The high-density linkage map QTLs reported in this study will be useful for molecular breeding of winged beans.

Dual-Purpose of the Winged Bean (Psophocarpus tetragonolobus (L.) DC.), the Neglected Tropical Legume, Based on Pod and Tuber Yields.

Winged beans (Psophocarpus tetragonolobus (L.) DC.) are grown as a vegetable legume crop in Thailand. All parts of the plant, including pods, seeds, leaves, flowers, and tubers are edible and are rich in protein and nutrients. Although the major consumption of winged bean is based on pod and tuber yields, only the people of Myanmar and Indonesia utilize winged bean tubers as food materials. The usefulness of the winged bean as an alternative crop for staple food and feed can shed some light on the impact of winged bean. Therefore, the evaluation of the dual purpose of the winged bean based on pod tuber yields is the objective of this study. In this study, ten-winged bean accessions-six accessions obtained from introduced sources and four accessions obtained from local Thai varieties-were laid out in randomized complete block design (RCBD) with three replications at the Agronomy Field Crop Station, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand from September 2019 to April 2020 and from October 2020 to April 2021. Data, including total pod weight, number of pods, pod length, 10-pod weight, and tuber weight were recorded, and the proximate nutrient and mineral contents in the tubers were also determined. The results revealed that the principal effects of year (Y) and genotype (G) were significant for total pod weight and the number of pods. Moreover, the Y × G interactions were principal effects upon the total pod weights and tuber weights. The results indicated that superior genotype and appropriate environmental conditions are key elements in successful winged bean production for both pod and tuber yields. The winged bean accessions W099 and W018 were consistent in both experimental years for pod and tuber yields at 23.6 and 18.36 T/ha and 15.20 and 15.5 T/ha, respectively. Each accession also proved high in tuber protein content at 20.92% and 21.04%, respectively, as well as significant in fiber, energy, and minerals. The results suggest that the winged bean accessions W099 and W018 can be used for dual-purpose winged bean production in Thailand.

Study of the Antioxidant and Anti-Inflammatory Properties of the Biological Extracts of Psophocarpus tetragonolobus Using Two Extraction Methods.

Psophocarpus tetragonolobus has long been used in traditional medicine and cuisine. In this study, Psophocarpus tetragonolobus extracts were isolated by maceration and ultrasound-assisted extraction and were evaluated for their antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The obtained results show that both extracts (maceration and ultrasound) were rich in bioactive molecules and exerted substantial antioxidant and anti-inflammatory effects. The P. tetragonolobus extracts' treatment in LPS-stimulated RAW264.7 macrophages resulted in a significant downregulation of the expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß mRNA. In addition, the P. tetragonolobus extracts' treatment attenuated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. Our observations indicate that there is no significant difference between the two studied extracts of P. tetragonolobus in terms of biological properties (specifically, antioxidant and anti-inflammatory effects. Regardless of the extraction method, P. tetragonolobus could be used for treating diseases related to oxidative stress and inflammatory reactions.

Psophocarpus tetragonolobus: An Underused Species with Multiple Potential Uses.

Natural products, particularly those extracted from plants, have been used as therapy for different diseases for thousands of years. The first written records on the plants used in natural medicine, referred to as "medicinal plants", go back to about 2600 BC. A thorough and complete understanding of medicinal plants encompasses a multiplex of overlapping and integrated sciences such as botany, pharmacognosy, chemistry, enzymology and genetics. Psophocarpus tetragonolobus, a member of Fabaceae family also called winged bean, is a perennial herbaceous plant characterized by its tuberous roots and its winged pod twinning and a perennial legume rich in proteins, oils, vitamins and carbohydrates. Besides nutrients, winged bean also contains bioactive compounds that have therapeutic activities like anti-oxidant, anti-inflammatory, antinociceptive, antibacterial, antifungal, antiproliferative and cytotoxic activity, a few of which already been reported. This plant can also be used as a medicinal plant for future benefits. With this concept in mind, the present review is designed to shed the light on the interests in the various phytochemicals and pharmacological pharmacognostical aspects of Psophocarpus tetragonolobus.

Effects of shading on the growth, development and yield of winged bean (Psophocarpus tetragonolobus) / Efeitos do sombreamento no crescimento, desenvolvimento e produtividade do feijão alado (Psophocarpus tetragonolobus)

ABSTRACT: An experiment was conducted to investigate the effects of different shading regimes [i.e., 60% (heavy), 30% (moderate), and 0% (control)] on 25 traits associated with the morphological features, photosynthetic gas exchange and agronomic characteristics of winged bean (Psophocarpus tetragonolobus), an underutilized protein-rich legume from the tropics. Collectively, 80% of the studied variables displayed significant differences (P<0.05) between at least two shade treatments. Shading generally showed most pronounced effect on the physiological traits of the legume, whereby the stomatal conductance, photosynthetic and transpiration rate differed significantly among plants for all treatments. The non-shaded plants were observed to have superior growth and physiological responses than the shaded plants. Interestingly, the moderately shaded plants exhibited the highest yield per plant, which significantly differed from the non-shaded and heavily shaded plants. This indicated that winged bean can adapt to partial canopy cover, making it a potential nitrogen-fixing cash crop which can be planted together with fruit or oil trees in commercial plantations.

RESUMO: Foi conduzido um experimento para investigar os efeitos de diferentes regimes de sombreamento [60% (pesado), 30% (moderado) e 0% (controle)] em 25 características associadas às características morfológicas, trocas gasosas fotossintéticas e características agronômicas de feijão alado (Psophocarpus tetragonolobus), leguminosa subutilizada rica em proteínas dos trópicos. Coletivamente, 80% das variáveis ​​estudadas apresentaram diferenças significativas (P<0,05) entre pelo menos dois tratamentos à sombra. O sombreamento mostrou efeito mais pronunciado nas características fisiológicas da leguminosa, pelo que a condutância estomática, a taxa fotossintética e a transpiração diferiram significativamente entre as plantas em todos os tratamentos. Observou-se que as plantas não sombreadas apresentaram crescimento e respostas fisiológicas superiores às plantas sombreadas. Curiosamente, as plantas moderadamente sombreadas exibiram o maior rendimento por planta, que diferiu significativamente das plantas não sombreadas e fortemente das sombreadas. Isso indica que o feijão alado pode se adaptar a cobertura parcial do dossel, tornando-o uma cultura potencial de fixação de nitrogênio que pode ser plantada em conjunto com árvores frutíferas ou oleaginosas em plantações comerciais.

Winged bean (Psophocarpus tetragonolobus (L.) DC.) for food and nutritional security: synthesis of past research and future direction.

MAIN CONCLUSION: Winged bean is popularly known as "One Species Supermarket" for its nutrient-dense green pods, immature seeds, tubers, leaves, and mature seeds. This underutilised crop has potential beneficial traits related to its biological nitrogen-fixation to support low-input farming. Drawing from past knowledge, and based on current technologies, we propose a roadmap for research and development of winged bean for sustainable food systems. Reliance on a handful of "major" crops has led to decreased diversity in crop species, agricultural systems and human diets. To reverse this trend, we need to encourage the greater use of minor, "orphan", underutilised species. These could contribute to an increase in crop diversity within agricultural systems, to improve human diets, and to support more sustainable and resilient food production systems. Among these underutilised species, winged bean (Psophocarpus tetragonolobus) has long been proposed as a crop for expanded use particularly in the humid tropics. It is an herbaceous perennial legume of equatorial environments and has been identified as a rich source of protein, with most parts of the plant being edible when appropriately prepared. However, to date, limited progress in structured improvement programmes has restricted the expansion of winged bean beyond its traditional confines. In this paper, we discuss the reasons for this and recommend approaches for better use of its genetic resources and related Psophocarpus species in developing improved varieties. We review studies on the growth, phenology, nodulation and nitrogen-fixation activity, breeding programmes, and molecular analyses. We then discuss prospects for the crop based on the greater understanding that these studies have provided and considering modern plant-breeding technologies and approaches. We propose a more targeted and structured research approach to fulfil the potential of winged bean to contribute to food security.

Growth and Phytoremediation Efficiency of Winged Bean in Fluorene- and Pyrene-Contaminated Soil.

Winged bean is a tropical legume that has been reported to enhance polycyclic aromatic hydrocarbon (PAH) biodegradation in soil. However, there is insufficient information about the susceptibility of winged bean to PAH toxicity in long term study. In this study, winged bean was planted in soil contaminated with either fluorene (124.5 mg/kg) or pyrene (98.4 mg/kg) for 90 days. Plant growth parameters and PAH disappearances from soil were measured every 30 days. Neither fluorene nor pyrene led to decreased shoot and root length of winged bean and all the winged bean plants flowered on day 90. However, the chlorophyll b content in the leaves decreased since day 60 and further decreased significantly by day 90 when winged bean was grown in the presence of fluorene or pyrene. The presence of fluorene and pyrene led to reduced root nodule formation at 30 and 60 days. Despite the reduced chlorophyll b content and decreased number of root nodules, winged bean could enhance pyrene removal significantly on day 30 compared to unplanted soil. Subsequently, pyrene degradation in the unplanted soil caught up and there was no statistically significant difference between the two treatments at 60 or 90 days. Negligible amounts of PAHs were accumulated in the shoot and root tissues of winged bean. These results showed that winged bean can speed up the removal of high MW PAHs from contaminated soil and we conclude that this plant is suitable for PAH phytoremediation.

Origin and diversification of winged bean (Psophocarpus tetragonolobus (L.) DC.), a multipurpose underutilized legume.

PREMISE OF THE STUDY: For many crops, research into the origin and partitioning of genetic variation is limited and this can slow or prevent crop improvement programs. Many of these underutilized crops have traits that could be of benefit in a changing climate due to stress tolerance or nutritional properties. Winged bean (Psophocarpus tetragonolobus (L.) DC.) is one such crop. All parts of the plant can be eaten, from the roots to the seeds, and is high in protein as well as other micronutrients. The goal of our study was to identify the wild progenitor and analyze the partitioning of genetic variation in the crop. METHODS: We used molecular phylogenetic analyses (cpDNA and nuclear ITS sequencing) to resolve relationships between all species in the genus, and population genetics (utilizing microsatellites) to identify genetic clusters of winged bean accessions and compare this to geography. KEY RESULTS: We find that winged bean is genetically distinct from all other members of the genus. We also provide support for four groups of species in the genus, largely, but not completely, corresponding to the results of previous morphological analyses. Within winged bean, population genetic analysis using 10 polymorphic microsatellite markers suggests four genetic groups; however, there is little correspondence between the genetic variation and the geography of the accessions. CONCLUSIONS: The true wild progenitor of winged bean remains unknown (or is extinct). There has likely been large-scale cross-breeding, trade, and transport of winged bean and/or multiple origins of the crop.

A Review on Current Status and Future Prospects of Winged Bean (Psophocarpus tetragonolobus) in Tropical Agriculture.

Winged bean, Psophocarpus tetragonolobus (L.) DC., is analogous to soybean in yield and nutritional quality, proving a valuable alternative to soybean in tropical regions of the world. The presence of anti-nutritional factors and high costs associated with indeterminate plant habit have been major concerns in this crop. But occurrence of good genetic variability in germplasm collections offers precious resources for winged bean breeding. However, lack of germplasm characterization is hindering such efforts. From a genomic standpoint, winged bean has been little studied despite rapid advancement in legume genomics in the last decade. Exploiting modern genomics/breeding approaches for genetic resource characterization and the breeding of early maturing, high yielding, determinate varieties which are disease resistant and free of anti-nutritional factors along with developing consumer friendly value-added products of local significance are great challenges and opportunities in the future that would boost cultivation of winged bean in the tropics. We review past efforts and future prospects towards winged bean improvement.

Understanding the molecular basis of stability in Kunitz (STI) family of inhibitors in terms of a conserved core tryptophan residue: A theoretical investigation.

ß-trefoil is one of the superfolds among proteins. Important classes of proteins like Interleukins (ILs), FibroblastGrowth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Kunitz (STI) family of inhibitors of proteins possess a highly conserved and structurally important Trytophan 91 (W91) residue, which stitches the top layer of the barrel with the lid. In this article we have investigated the molecular insights of the involvement of this W91 residue in the stability and folding pathway of Kunitz (STI) family. Winged bean Chymotrypsin inhibitor (WCI), a member of Kunitz (STI) family was chosen as a model system for carrying out the work. Molecular dynamics (MD) simulations were run with a set of total six proteins, including wild type WCI (WT) & five mutants namely W91F, W91M, W91A, W91H and W91I. Among all of them the coordinates of four proteins were taken from their crystal structures deposited in the Protein Data Bank (PDB), where as the coordinates for the rest two was generated using in-silico modelling. Our results suggest that truly this W91 residue plays a determining role in stability and folding pathway of Kunitz (STI) family. The mutants are less stable and more susceptible to quicker unfolding at higher temperatures compared to the wild type WCI. These effects are most pronounced for the smallest mutants namely W91H and W91A, indicating more is the cavity created by mutation at W91 position more the proteins becomes unstable.

Development of Gene-Based SSR Markers in Winged Bean (Psophocarpus tetragonolobus (L.) DC.) for Diversity Assessment.

Winged bean (Psophocarpus tetragonolobus) is an herbaceous multipurpose legume grown in hot and humid countries as a pulse, vegetable (leaves and pods), or root tuber crop depending on local consumption preferences. In addition to its different nutrient-rich edible parts which could contribute to food and nutritional security, it is an efficient nitrogen fixer as a component of sustainable agricultural systems. Generating genetic resources and improved lines would help to accelerate the breeding improvement of this crop, as the lack of improved cultivars adapted to specific environments has been one of the limitations preventing wider use. A transcriptomic de novo assembly was constructed from four tissues: leaf, root, pod, and reproductive tissues from Malaysian accessions, comprising of 198,554 contigs with a N50 of 1462 bp. Of these, 138,958 (70.0%) could be annotated. Among 9682 genic simple sequence repeat (SSR) motifs identified (excluding monomer repeats), trinucleotide-repeats were the most abundant (4855), followed by di-nucleotide (4500) repeats. A total of 18 SSR markers targeting di- and tri-nucleotide repeats have been validated as polymorphic markers based on an initial assessment of nine genotypes originated from five countries. A cluster analysis revealed provisional clusters among this limited, yet diverse selection of germplasm. The developed assembly and validated genic SSRs in this study provide a foundation for a better understanding of the plant breeding system for the genetic improvement of winged bean.

Structure-function relationship of a bio-pesticidal trypsin/chymotrypsin inhibitor from winged bean.

Protease inhibitors are essential bio-molecules that serve as a model system for the study of protein structure and protease-protease inhibitor interaction. We here report a bi-functional serine protease inhibitor from winged bean (WBCTI) that completely retains its inhibitory property against trypsin and chymotrypsin even after heating at 70°C. Detailed circular dichroism and fluorescence studies at different temperatures, 30-90°C, have been performed to understand the reason behind thermal stability of the protein. On the basis of our results it appears that WBCTI maintains its canonical structure up to 70°C. Above that the heat induced conformational change becomes irreversible which causes aggregation followed by precipitation of the protein. Moreover, the activity and stability of the secondary structure are found to decrease drastically in presence of dithiothreitol indicating that the protein acquires additional stability for the occurrence of two disulfide bonds. In addition to the structural characterization, an important property of WBCTI against the polyphagous pest Helicoverpa armigera has been explored in present study. WBCTI has showed reasonable inhibition of the mid-gut proteases of H. armigera. In artificial feeding trial through addition of WBCTI in diet resulted in significant growth retardation, delayed pupae formation and higher mortality of H. armigera larvae.

Molecular and biological properties of an endornavirus infecting winged bean (Psophocarpus tetragonolobus).

A double-stranded RNA (dsRNA) of approximately 15 kbp was isolated from asymptomatic winged bean (Psophocarpus tetragonolobus) plants. The size of the dsRNA, together with results of RT-PCR testing, suggested that it was the replicative form of a plant endornavirus. Cloning, sequencing, and sequence analyses confirmed the endornavirus nature of the dsRNA. Conserved motifs typical for endornaviruses were identified and their amino acid sequences compared with those of selected endornaviruses. Phylogenetic analyses revealed a close relationship with Bell pepper endornavirus, Phaseolus vulgaris endornavirus 2, and Hot pepper endornavirus. The dsRNA was present in most P. tetragonolobus genotypes tested. The virus was provisionally named Winged bean endornavirus 1 (WBEV-1).

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of Psophocarpus tetragonolobus (L.) DC.

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L(-1) induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L(-1)) and IAA (0.2 mg L(-1)) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.

Preparation of bioactive peptides with high angiotensin converting enzyme inhibitory activity from winged bean [Psophocarpus tetragonolobus (L.) DC.] seed.

Winged bean [Psophocarpus tetragonolobus (L.) DC.] seed is a potential underexploited source of vegetable protein due to its high protein content. In the present work, undefatted and defatted winged bean seed hydrolysates, designated as UWBSH and DWBSH, respectively were produced separately by four proteolytic enzymes namely Flavourzyme, Alcalase, Bromelain, and Papain using pH-stat method in a batch reactor. Enzymatic hydrolysis was carried out over a period of 0.5 to 5 h. UWBSH and DWBSH produced were tested for their ACE inhibitory activity in relation to the hydrolysis time and degree of hydrolysis (DH). Maximum ACE inhibitory activity, both for UWBSH and DWBSH, were observed during 3 to 5 h of hydrolysis. Both, UWBSH (DH 91.84 %), and DWSBH (DH 18.72 %), produced by Papain at 5 h hydrolysis, exhibited exceptionally high ACE inhibitory activity with IC50 value 0.064 and 0.249 mg mL(-1), respectively. Besides, papain-produced UWBSH and DWBSH were further fractionated into three fractions based on molecular weight (UWBSH-I, <10 kDa; UWBSH-II, <5 kDa; UWBSH-III, <2 kDa) and (DWBSH-I, <10 kDa; DWBSH-II, <5 kDa; DWBSH-III, <2 kDa). UWBSH-III revealed the highest ACE inhibitory activity (IC50 0.003 mg mL(-1)) compared with DWBSH-III (IC50 0.130 mg mL(-1)). The results of the present investigation revealed that winged bean seed hydrolysates can be explored as a potential source of ACE inhibitory peptides suggesting their uses for physiological benefits as well as for other functional food applications.