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In the world of highly processed foods, special attention is drawn to the nutrient composition and safety of consumed food products. Foods fortified with probiotic bacteria confer beneficial effects on human health and are categorized as functional foods. The salubrious activities of probiotics include the synthesis of vital bioactives, prevention of inflammatory diseases, anticancerous, hypocholesterolemic, and antidiarrheal effects. Soy foods are exemplary delivery vehicles for probiotics and prebiotics and there are diverse strategies to enhance their functionality like employing mixed culture fermentation, engineering probiotics, and incorporating prebiotics in fermented soy foods. High potential is ascribed to the concurrent use of probiotics and prebiotics in one product, termed as "synbiotics," which implicates

Pearl millet is a nutrient dense and gluten free cereal, however it's flour remains underutilized due to the onset of rancidity during its storage. To the best of our knowledge, processing methods, which could significantly reduce the rancidity of the pearl millet flour during storage, are non-existent. In this study, pearl millet grains were subjected to a preliminary hydro-treatment (HT). Subsequently, the hydrated grain-wet flour have undergone individual and combined thermal treatments viz., hydrothermal (HTh) and thermal near infrared rays (thNIR). Effects of these thermal treatments on the biochemical process of hydrolytic and oxidative rancidity were analyzed in stored flour. A significant (p 0.05) decrease in the enzyme activities of lipase (47.8%), lipoxygenase (84.8%), peroxidase (98.1%) and polyphenol oxidase (100%) in HT-HTh-thNIR treated flour compared to the individual treatments was documented. Upon storage (90 days), decline of 67.84% and 66.4% of free fatty acid and peroxide contents were observed in flour under HT-HTh-thNIR treatment without altering starch and protein digestibility properties. HT-HTh treated flour exhibited the highest (7.6%) rapidly digestible starch, decreased viscosity and increased starch digestibility (67.17%). FTIR analysis of HT-HTh treated flour divulged destabilization of short-range ordered crystalline structure and altered protein structures with decreased in vitro digestibility of protein. Overall, these results demonstrated the effectiveness of combined thermal treatment of HT-HTh-thNIR in reducing rancidity and preserving the functional properties of the stored flour.

Starch-sugar homeostasis and starch molecular configuration regulates the dynamics of starch digestibility which result in sweet sensory perception and eliciting glycemic response, which has been measured in vitro as inherent glycemic potential (IGP). The objective of the research was to understand the key determinants of IGP as well as sweetness in different Pearl millet (PM) genotypes. To understand the intricate balance between starch and sugar, total starch content (TSC) and total soluble sugars (TSS) were evaluated. Higher concentrations of TSC (67.8%), TSS (2.75%), glucose (0.78%) and sucrose (1.68%) were found in Jafarabadi Bajra. Considering the role of compact molecular configuration of starch towards digestibility, X-ray powder diffraction (XRD) analysis was performed. A-type crystallinity with crystallinity degree (CD %) ranged from 53.53-62.63% among different genotypes, where the least CD% (53.53%) was found in Jafarabadi Bajra. In vitro starch hydrolyzation kinetics carried out to determine IGP, revealed a maximum of 77.05% IGP with minimum 1.42% resistant starch (RS) in Jafarabadi Bajra. Overall our results suggest higher sweet sensory perception of Jafarabadi Bajra which is contributed by the matrix composition with least molecular compactness of starch. Also, the interdependence among starch quality parameters; CD%, IGP, RS and amylose has also been discussed.

Soybean (Glycine max (L.) Merr) is a nutrient-rich crop with limited consumer acceptability because of the presence of off-flavour. Owing to the ubiquity of isoflavones in soybean, their inherent antioxidant potential, and their inhibitory effect on lipoxygenase activity, their sensorial attribute in mitigating off-flavour is currently being envisaged. We estimated the content and composition of isoflavones in 17 soybean cultivars and established the correlation between isoflavone profile and lipid peroxidation, which was measured in terms of thiobarbituric acid (TBA) number and carbonyl value. The results showed a significant negative correlation of these parameters with the total isoflavone content and genistein: daidzein ratio. In addition, expression analysis of a key gene involved in isoflavone biosynthesis, i.e., isoflavone synthase (IFS1 and IFS2), in contrasting soybean genotypes during seed development revealed a higher expression of both isoforms in Bragg (high isoflavone) compared to PUSA 40 (low isoflavone) at different stages of seed development accompanied by more isoflavone accumulation. The cloning and in silico analysis of IFS isoforms revealed a more significant interaction with other biosynthesis enzymes by IFS2, indicating its more substantial role in isoflavone synthesis. IFS2 overexpression can thus be a valuable target to increase isoflavone biosynthesis, thereby scavenging off-flavour.

Soybean (Glycine max (L.) Merr) is a nutrient-rich crop but has limited consumer acceptability because of the presence of off-flavour. Owing to the ubiquity of isoflavones in soybean, their inherent antioxidant potential, and inhibitory effect on lipoxygenase activity, their sensorial attribute in mitigating off-flavour is currently being envisaged. We estimated the content and composition of isoflavones in 17 soybean cultivars and established the correlation between isoflavone profile and lipid peroxidation, which was measured in terms of Thiobarbituric acid (TBA) number and carbonyl value. The results showed a significant negative correlation of these parameters with the total isoflavone content and genistein: daidzein ratio. Besides, expression analysis of a key gene involved in isoflavone biosynthesis i.e., isoflavone synthase (IFS1 and IFS2) in contrasting soybean genotypes during seed development revealed a higher expression of both isoforms in Bragg (High isoflavone) as compared to PUSA 40 (Low isoflavone) at different stages of seed development accompanied by more isoflavone accumulation. The cloning and in silico analysis of IFS isoforms revealed a more significant interaction with other biosynthesis enzymes by the IFS2, indicating its more substantial role in isoflavone synthesis. IFS2 overexpression thus can be a valuable target to increase isoflavone biosynthesis, thereby scavenging off-flavour.

Soybean oil has high contents of polyunsaturated fatty acids and hence susceptible to deterioration due to the presence of lipase and lipoxygenase (LOX) enzymes. These enzymes cause the severe impact in the reduction of shelf life. Therefore it is indispensable to adopt a stabilization step to increase antioxidant levels and to decrease the activity of lipase and LOX enzymes. To carry out this study soybean varieties were irradiated by low gamma doses radiation (0.5 kGy and 1.0 kGy) and oil was stored up to 60 days at 40 °C with 45% relative humidity. Results indicated the lowest activities of lipase (20.9%) and LOX (17.1%) at 60 days of storage respectively and also observed significantly low hydrolytic (22.6%) and oxidative rancidity (64%) respectively in Bragg oil under the condition of 0.5 kGy gamma irradiation. Total antioxidants were found to be increased including lipid soluble antioxidants like tocopherols and also found low level of reactive oxygen species (ROS) deposition in Bragg seeds irradiated with 0.5 kGy which suggests that low dose of gamma irradiation is highly effective in enhancing the oil stability and also in retaining antioxidants.

Millets are known for its nutritional excellence because of its high nutrient density. Among all the millets, pearl millet is considered the most important nutricereals, having 3–8% lipids. Rancidity or off-odour development in flour is one of the major limiting factors in the popularization of pearl millet all over the world. In the present investigation, we have evaluated the flour (freshly milled and 10 days after milling) of diverse genotypes of pearl millet for their hydrolytic and oxidative degradation of lipids using different biochemical parameters such as comprehensive acid value (CAV: the number of milligrammes of potassium hydroxide required to neutralize the free fatty acids present in 1 g of flour), comprehensive peroxide value (CPV: determination of the primary oxidation product i.e. hydroperoxides in the flour) and activities of lipase and lipoxygenase (LOX). We have classified pearl millet genotypes into low, medium and high rancid groups and developed a rancidity matrix (RM) with 3 groups and 6 classes; the matrix was validated using 93 diverse pearl millet genotypes. RM will be of great help to the pearl millet breeders for developing low rancid pearl millet lines.

More than as a phenotypic marker for breeding, seed coat colour of soybean is gaining momentum as a nutraceutical marker owing to the multitude of medicinal effects provided by anthocyanins. The acute obstacle for large scale phenotyping is a rapid, reliable and accurate quantification which simultaneously determines various anthocyanins and hence, in this study, the modified method efficiently separated cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G) and petunidin-3-glucoside (Pt3G) forms by eluting through a RP-C18 column with an optimized isocratic mobile phase containing 18% solvent B (0.4% trifluoro acetic acid in acetonitrile) in solvent A (0.4% trifluoro acetic acid in water). The elution profile of anthocyanins were C3G > D3G > Pt3G, with C3G as the predominant (~ 85%) form. The modified method was validated in terms of linearity (R2 = 0.998), low limit of detection (LOD = 5.8 μg ml−1), limit of quantification (LOQ = 22.25 μg ml−1), precision, repeatability, stability and recovery. C3G dynamics was found increased in a linear trend from 30DAF to later developing stages until maturity. The investigation on characterization of exotic soybean genotypes revealed that, maximum C3G content of 4.9 mg g−1 was in UPSL496 and the least in EC471921 (3.56 mg g−1). There was a positive correlation observed among all the variables, like monomeric anthocyanin content (MAC), C3G, D3G and Pt3G. Clustering and heat map analysis information on this efficient method can be used for future research for germ plasm evaluation and for developing nutritionally C3G enriched high yielding varieties.

The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9) is simple and highly efficient technology applied to functional studies of genes and genetic crop improvement. In this study, we have demonstrated the utility of green fluorescent protein (GFP) marker to detect the targeting efficiency of gRNAs. As a proof of concept, Glycine max De-Etiolated 1 (GmDET1) gene was chosen and tagged with GFP to rapidly analyze genome editing efficiency of gRNAs. Results showed weaker GFP fluorescence signal in the N. benthamiana leaves co-infiltrated with GmDET1-GFP overexpression (OE) + DET1 gRNA1 constructs as compared to the stronger GFP florescence signal in the leaves co-infiltrated with DET1 gRNA2 and gRNA3 constructs, thus indicating the highest of DET1 gRNA1. These results were further confirmed by the detection of the mutation frequencies through T7 endonuclease (T7E1) assay and sequencing; the highest mutation rate of 38.46% in GmDET1 targeted by DET1 gRNA1 to that of DET1 gRNA2 (7.69%) and gRNA3 (15.38%) was observed. Thus our studies showed “GFP tagging” as the most reliable and rapid method-one can apply to minimize the generation of non-edited transgenic plants resulting from inefficient gRNAs.

Millets are recently being recognized as emerging food ingredients with multifaceted applications. Whole grain flours made from millets, exhibit diverse chemical compositions, starch digestibility and physicochemical properties. A food matrix can be viewed as a section of food microstructure, commonly coinciding with a physical spatial domain that interacts or imparts specific functionalities to a particular food constituent. The complex millet-based food matrices can help individuals to attain nutritional benefits due to the intricate and unique digestive properties of these foods. This review helps to fundamentally understand the binary and ternary interactions of millet-based foods. Nutritional bioavailability and bioaccessibility are also discussed based on additive, synergistic, masking, the antagonistic or neutralizing effect of different food matrix components on each other and the surrounding medium. The molecular basis of these interactions and their effect on important functional attributes like starch retrogradation, gelling, pasting, water, and oil holding capacity is also discussed.

Transient gene expression utilizing syringe mediated agro infiltration offers a simple yet efficient technique for various transgenic applications. Although soybean is commonly used as a model plant for functional genomic studies, till date there have been no reports available on a high throughput agro infiltration protocol for transient gene expression. In the present study, we developed a simple transient expression system, named Agrobacterium-mediated disc assay for transient expression (AGRO DATE) in mature soybean leaves involving the use of an optimized infiltration buffer [10 mM 2-(N-morpholino) ethane sulfonic acid sodium salt, 10 mM MgCl2, 0.2 mM acetosyringone, 400 mg L−1, l-cysteine, 0.5 mM dithiothreitol and 0.01% Tween 20] under vacuum using a needle-less syringe. This not only limits the genetic pollution but can also be conducted without the intervention of any specialized equipments. Model protein β-glucuronidase (GUS) was used for optimizing various parameters and the synergized composition delivered 58% transformation efficiency within 4 days of infiltration. We demonstrated the versatile applicability of the method for examining reporter genes (GUS and bar), over expression (GmIPK1) and antisense suppression of GmMIPS in soybean. In addition, AGRODATE offers a viable approach for application of agro infiltration in other recalcitrant plant species and may become a useful tool for the research community.

Despite the significant importance of soybean isoflavone, the regulatory mechanism of miRNAs during its biosynthesis is highly unexplored. In the present work, nine existing miRNAs along with their ten corresponding target genes were identified and validated in soybean for their possible role during isoflavonoid biosynthesis and accumulation. Temporal expression analysis at four key stages of seed development (35, 45, 55 and 65DAF) of all the miRNA-target pairs showed varying degree of differential accumulation in two soybean genotypes (NRC37: high isoflavone; and NRC7: low isoflavone). Differential expression of MYB65-Gma-miR159, MYB96-Gma-miRNA1534, MYB176-Gma-miRNA5030, SPL9-Gma-miRNA156, TCP3, TCP4-Gma-miRNA319, WD40-Gma-miRNA162, UDP-glucose: flavonoid 3-O-glucosyltransferase-Gma-miRNA396, and CHI3-Gma-miRNA5434 showed an important relationship with their targets in both the soybean genotypes across all the stages. Therefore, the finding of the present work would certainly increase our understanding of molecular regulation of isoflavone biosynthetic pathway mediated by the miRNA which would guide molecular breeder to develop isoflavone rich soybean cultivars.

The biochemical basis behind the preferential presence and action of GmITPKs (myo-inositol—1, 3, 4 tris phosphate 5/6 kinases) in regulating the phosphorous flux towards phytate synthesis in soybean is an important, still unresolved question. In this study, context specific expression of GmITPKs in developing seeds was analysed, which found to be maximum at early stages (0–4 mm); while isozyme specificity was observed with varied dynamics till maturity. It is commonly believed that similarity at sequence level will infer similarity in structure as well, but the robust correlation between sequence and structure has not yet been proved. Hence full length coding sequences of GmITPKs were cloned and their sequence comparison revealed a low identity score of about 50% among the isoforms. Creative computational tools were employed to predict and comprehend the structural attributes and to explore the inverse correlation between the structural and sequence similarities of GmITPKs. Homology modeling of GmITPKs, their refinement and superposition validated the functional isoformic nature, with 92–97% similarity in spatial configuration. Ligand docking and energy computations showed ADP, IP6 and IP5 as possible ligands, and interactions were studied using docking and simulation. The homology models of GmITPKs were used as the starting point for nanosecond-duration molecular dynamics and were found to be stable during 20 ns simulation period. Existence of situation-specific functional basis for this isoform multiplicity, at structural and expression levels revealed to be a preferred regulatory mechanism to adapt to the metabolic fluctuations in inositol pathway during seed development.

The present study compares three methods viz. microwave assisted extraction (MAE), ultrasonic-assisted extraction (UAE) and conventional solvent extraction (CSE) for extraction of phenolic compounds from black carrot pomace (BCP). BCP is the major by-product generated during processing and poses big disposal problem. Box–Behnken design using response surface methodology was employed to investigate and optimize the MAE of phenolics, antioxidant activity and colour density from BCP. The conditions for maximum recovery of polyphenolics were: microwave power (348.07 W), extraction time (9.8 min), solvent–solid ratio (19.3 mL/g) and ethanol concentration (19.8%). Under these conditions, the extract contained total phenolic content of 264.9 ± 10.02 mg gallic acid equivalents (GAE)/100 mL, antioxidant capacity (AOC) of 13.14 ± 1.05 µmol Trolox equivalents (TE)/mL and colour density of 68.63 ± 5.40 units. The total anthocyanin content at optimized condition was 753.40 ± 31.6 mg/L with low % polymeric colour of 7.40 ± 0.42. At optimized conditions, MAE yielded higher colour density (68.63 ± 5.40), polyphenolic content (264.9 ± 10.025 mg GAE/100 mL) and AOC (13.14 ± 1.05 µmol TE/mL) in a short time as compared to UAE and CSE. Overall results clearly indicate that MAE is the best suited method for extraction in comparison to UAE and CSE. The phenolic rich extract can be used as an effective functional ingredient in foods. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-018-03566-9) contains supplementary material, which is available to authorized users.

Anthocyanin-rich extracts from black carrots are being considered as a candidate replacer for the red colour in processed foods. The present investigation optimizes the extraction of anthocyanins with high phenolic content and low degradation parameters from black carrots using a cellulolytic multi-enzyme preparation known as Viscozyme. The optimized conditions for Viscozyme using a Box-Behnken design (BBD) of response surface methodology (RSM) were as follow: temperature (50.2 ◦C), extraction time (58.4 min) and enzyme concentration (0.20%). The predicted value of anthocyanins content was 1380 mg/L, which was near to the optimized experimental value of 1375 mg/L. The extracted anthocyanins based on above mentioned conditions exhibited the lowest degradation parameters such as degradation index (DI) of (0.86), browning index of (BI) (1.31) and were characterized with cyanidin 3-sinapoylxylosylglucosylgalactoside as being the most abundant. The findings clearly reveal that Viscozyme-assisted extraction (VAE) is the best approach for extracting superior quality extracts from black carrots with high anthocyanin and other phenolic component concentrations.

The ATP-binding cassette (ABC) transporters belong to a large protein family predominantly present in diverse species. ABC transporters are driven by ATP hydrolysis and can act as exporters as well as importers. These proteins are localized in the membranes of chloroplasts, mitochondria, peroxisomes and vacuoles. ABC proteins are involved in regulating diverse biological processes in plants, such as growth, development, uptake of nutrients, tolerance to biotic and abiotic stresses, tolerance to metal toxicity, stomatal closure, shape and size of grains, protection of pollens, transport of phytohormones, etc. In mitochondria and chloroplast, the iron metabolism and its transport across the membrane are mediated by ABC transporters. Tonoplast-localized ABC transporters are involved in internal detoxification of metal ion; thus protecting against the DNA impairment and maintaining cell growth. ABC transporters are involved in the transport of secondary metabolites inside the cells. Microorganisms also engage a large number of ABC transporters to import and expel substrates decisive for their pathogenesis. ABC transporters also suppress the seed embryonic growth until favorable conditions come. This review aims at giving insights on ABC transporters, their evolution, structure, functions and roles in different biological processes for helping the terrestrial plants to survive under adverse environmental conditions. These specialized plant membrane transporters ensure a sustainable economic yield and high-quality products, especially under unfavorable conditions of growth. These transporters can be suitably manipulated to develop 'Plants for the Future'.

Low phytate soybeans are desirable both from a nutritional and economic standpoint. Inositol 1, 3, 4, 5, 6-pentakisphosphate 2-kinase (IPK1), optimizes the metabolic flux of phytate generation in soybean and thus shows much promise as a likely candidate for pathway regulation. In the present study, the differential spatial and temporal expression profiling of GmIpk1 and its two homologs Glyma06g03310 and Glyma04g03310 were carried out in Glycine max L. var Pusa 9712 revealing the early stages of seed development to be the potential target for gene manipulation. NCBI databank was screened using BLASTp to retrieve 32 plant IPK1 sequences showing high homology to GmIPK1 and its homologs. Bio-computational tools were employed to predict the protein’s properties, conserved domains, and secondary structures. Using state-of-the-art in silico physicochemical approach, the three-dimensional (3D) GmIPK1 protein model (PMD ID—PM0079931), was developed based on Arabidopsis thaliana (PDB ID: 4AQK). Superimposition of 4AQK and best model of GmIPK1 revealed that the GmIPK1 aligned well and shows a sequence identity score of 54.32% with 4AQK and a low RMSD of 0.163 nm and almost similar structural features. The modeled structure was further refined considering the stereochemical geometry, energy and packing environment between the model and the template along with validation of its intrinsic dynamics. Molecular dynamics simulation studies of GmIPK1 were carried out to obtain structural insights and to understand the interactive behavior of this enzyme with ligands ADP and IP(6). The results of this study provide some fundamental knowledge on the distinct mechanistic step performed by the key residues to elucidate the structure–function relationship of GmIPK1, as an initiative towards engineering “low phytate soybean”. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02260-y) contains supplementary material, which is available to authorized users.

Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc—the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of γ-TMT1 and γ-TMT3 were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, Glycine max-γ-TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in Nicotiana benthamiana using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress.

In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD600) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L−1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L−1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after s-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.

Application of extracted black carrot anthocyanins (BCA) and black soybean anthocyanins (BSA) as colour enhancers was demonstrated by copigmenting with heat sensitive anthocyanins from plum, pomegranate, strawberry and jamun. Improved pigment stability was confirmed by thermal stability, chromacity studies and hyperchromic and bathochromic shifts. BCA (2%, 500 mg/L) caused largest hyperchromic shift in plum whereas maximum bathochromic shift (18–20 nm) was observed in strawberry. BSA (2%, 500 mg/L) caused largest hyperchromic shift in pomegranate whereas maximum bathochromic shift (22 nm) was observed in plum. Copigmented plum, jamun, strawberry and pomegranate juices copigmented with BCA achieved high half-life (t1/2) and lower rate constant (k) values. Copigmenting anthocyanins with other juice is an effective strategy to impart high thermal stability, improve colour retention and antioxidant activity.

Radiation processing of soybean, varying in seed coat colour, was carried out at dose levels of 0.25, 0.5 and 1 kGy to evaluate their potential anti-proliferative and cytoprotective effects in an in vitro cell culture system. Irradiated and control black (Kalitur) and yellow (DS9712) soybean extracts were characterized in terms of total phenolics, flavonoids and anthocyanins, especially cyanidin-3-glucoside (C3G). Using an epithelial cell line, BEAS-2B the potential cytoprotective effects of soybean extracts were evaluated in terms of intracellular ROS levels and cell viability. The most relevant scavenging effect was found in Kalitur, with 78% decrease in ROS, which well correlated with a 33% increase in C3G after a 1 kGy dose. Results evidenced a correspondence between in vitro antioxidant activity and a potential health property of black soybean extracts, exemplifying the nutraceutical role of C3G. To our knowledge this study is the first report validating the cytoprotective effects of irradiated black soybean extracts.

Phytic acid (PA) is implicative in a spectrum of biochemical and physiological processes involved in plant stress response. Inositol 1,3,4, Tris phosphate 5/6 kinase (ITPK), a polyphosphate kinase that converts Inositol 1,3,4 trisphosphate to Inositol 1,3,4,5/6 tetra phosphate, averting the inositol phosphate pool towards PA biosynthesis, is a key regulator that exists in four different isoforms in soybean. In the present study, in-silico analysis of the promoter region of ITPKs was done and among the four isoforms, promoter region of GmITPK2 showed the presence of two MYB binding elements for drought inducibility and one for ABA response. Expression profiling through qRT-PCR under drought and salinity stress showed higher expression of GmITPK2 isoform compared to the other members of the family. The study revealed GmITPK2 as an early dehydration responsive gene which is also induced by dehydration and exogenous treatment with ABA. To evaluate the osmo-protective role of GmITPK2, attempts were made to assess the bacterial growth on Luria Broth media containing 200 mM NaCl, 16% PEG and 100 μM ABA, individually. The transformed E. coli BL21 (DE3) cells harbouring the GmITPK2 gene depicted better growth on the media compared to the bacterial cells containing the vector alone. Similarly, the growth of the transformed cells in the liquid media containing 200 mM NaCl, 16% PEG and 100 μM ABA showed higher absorbance at 600 nm compared to control, at different time intervals. The GmITPK2 recombinant E. coli cells showing tolerance to drought and salinity thus demonstrated the functional redundancy of the gene across taxa. The purity and specificity of the recombinant protein was assessed and confirmed through PAGE showing a band of ∼35 kDa on western blotting using Anti- Penta His- HRP conjugate antibody. To the best of our knowledge, the present study is the first report exemplifying the role of GmITPK2 isoform in drought and salinity tolerance in soybean.

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74-87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded β sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni2+-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules.

The nutritional composition of 87 diverse Pearl millet (Pennisetum glaucum (L.) R. Br.) germplasm including landraces and commercial varieties was assessed through standard protocols. The results indicated a substantial variability in total carbohydrates [starch (50.37–63.25), amylose (19.26–27.90), sucrose (0.58–1.53), glucose (0.32–0.75), resistant starch (RS) (1.49–3.52), total soluble sugars (TSS) (1.53–3.22), expressed as g/100 g], protein (8.07–18.15 g/100 g), total dietary fibre (TDF) (7.68–16.18 g/100 g), lipids and fatty acids [total lipid (5.24–9.99), palmitic (20.30–32.49), linoleic (32.11–46.91), oleic (21.99–33.43) and stearic acid (3.28–7.91) expressed as g/100 g], antinutritional factors [phytic acid (0.54–1.43 g/100 g) and raffinose family oligosaccharides (RFOs) (0.27–2.08 mmol/100 g)], phenols (0.04–0.21 g/100 g), and minerals. Multivariate analysis using hierarchical clustering analysis (HCA), and principal component analysis (PCA) was used to decipher the diversity of these attributes across germplasm. Multivariate data analysis (MVDA) can be applied for deciphering the differences/ similarities between multiple nutritional attributes, sample types or for projecting the object in a two/ three-dimensional factor-plane, determined based on various distinct characteristics. HCA revealed that Cluster I, II and III showed higher content of amylose, starch, moisture, cluster III had higher lipid content. cluster I, II, III and IV showed higher RS. Cluster II and III had higher TSS, cluster III showed higher sucrose content. Cluster V and VII were indicated by higher glucose and protein content. Cluster II, III, IV and VI showed phytic acid content and cluster III showed higher mineral content. The germplasm displayed distinct regiospecific variations in their nutritional content. Those derived from Gujarat, Maharashtra and Uttarakhand showed higher protein content. Those derived from Haryana, Karnataka, New Delhi, Punjab, Tamil Nadu and Uttar Pradesh showed high carbohydrates. Those from New Delhi, Punjab, Tamil Nadu and Uttarakhand showed high Iron content and high copper was found in germplasm from Maharashtra, Punjab and Tamil Nadu. High zinc was found in germplasms from Maharashtra, Punjab, Tamil Nadu and Uttar Pradesh. More calcium was found in germplasm from Maharashtra, Punjab, New Delhi, and Tamil Nadu. The analysis can form the basis for the commercialization and utilization of pearl millet using efficacious breeding strategies.

Soybeans are known for its good source of protein (40%), oil (20%) and also serve as a source of nutraceutical compounds including tocopherols (toc). To know the molecular basis of differential α-toc accumulation in two contrasting soybean genotypes: DS74 (low α-toc - 1.36 μg/g and total-toc -29.72 μg/g) and Bragg (high α-toc - 10.48 μg/g and total-toc 178.91 μg/g), the analysis of γ-TMT3 promoter activity and its methylation patterns were carried out. The sequencing results revealed nucleotide variation between Bragg:γ-TMT3-P and DS74:γ-TMT3-P, however none of the variations were found in core-promoter region or in cis-elements. The histochemical GUS assay revealed higher promoter activity of Bragg:γ-TMT3-P than that of DS74:γ-TMT3-P and correlated with significantly higher and lower (P 0.05) expression of γ-TMT3 gene respectively. To know the molecular basis of differential accumulation of α-toc in these contrasting soybean genotypes, the DNA methylation pattern of γ-TMT3 gene body and its promoter was studied in both varieties. The results showed higher percentage (62.5%) of methylation in DS74:γ-TMT3-P than in Bragg:γ-TMT3-P (50%). Out of all the methylation sites in the promoter region, one of methylation site was found at CAAT box (-190 bp) of DS74:γ-TMT3-P. Further gene body methylation patterns revealed lowest % (40%) of CG methylation in DS74:γ-TMT3 gene as compared to Bragg:γ-TMT3 (64.2%). Thus our study revealed that, expression of γ-TMT3 gene was influenced by its promoter activity and methylation patterns in cis-elements of γ-TMT3 promoter and gene body. This study will help us to understand the possible role of methylation and promoter activity in determining the α-toc content in soybean seeds.

Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc—the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of γ-TMT1 and γ-TMT3 were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, Glycine max-γ-TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in Nicotiana benthamiana using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-2086-9) contains supplementary material, which is available to authorized users.

The present study compares three methods viz. microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE) and conventional solvent extraction (CSE) for extraction of polyphenolic compounds from Black Soybean Seed coat (BSSC). Box-Behnken design using response surface methodology (RSM) was employed to investigate and optimize the MAE and EAE for maximum bioactive content, antioxidant activity, colour density and minimum degradation parameters from BSSC. Optimized MAE conditions for BSSC were: microwave power of 569.46 W, extraction time of 262.54 s, solvent to solid ratio of 40:1 and ethanol concentration (59.99). The predicted anthocyanin content was 5021.47 mg/l, close to experimental optimized value of 5094.9 mg/l with minimum values of degradation parameters viz., Polymeric Colour (PC) (0.131 ± 0.01), Browning Index (BI) (0.202 ± 0.02) and Degradation Index (DI) (0.140 ± 0.02). Overall results clearly indicate that MAE is the best suited method for extraction in comparison to EAE and CSE. The phenolic rich extract can be used as an effective functional ingredient in foods.

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60–80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Consumption of soybean is dependent on its protein quality and texture which is influenced by protein modification. In the present study, modifications of soybean proteins were investigated by chemical analysis. Irradiation dose of 2.0 kGy resulted in reduced turbidity, surface hydrophobicity and protein oxidation, but caused increase in protein solubility and sulfhydryl contents in both the genotypes. Free phenolics, DPPH and FRAP activites in all the genotypes increased significantly at 2.0 kGy and declined at 5.0 kGy with the exception of DPPH. However, lipoxygenase and bound phenolics decreased with the increase doses of gamma irradiation in both the genotypes tested. To the best of our knowledge this study is the virgin report exploring the use of gamma irradiation for improvement of soybean protein stability, quality and its bioavailability by reducing protein oxidation& modification and/or by enhancing the total inherent antioxidant capacity.

Defatting soybean by sophisticated oil extraction method utilising supercritical CO2 resulted in a significant decrease in the residual phospholipids (PLs) compared with soymeal obtained by conventional cold percolation method utilising hexane as the extraction solvent. Interestingly, the levels of residual PLs showed a proportionate relationship with thiobarbituric acid (TBA) number, an indicator of lipid peroxidation responsible for off-flavour generation. Furthermore, two oleosins (18 and 24 kDa) were isolated from the oil bodies extracted from soybean seeds and positively characterised for phospholipase A2 (PLA2) activity, suggesting their plausible involvement in off-flavour generation in soymeal. The treatment of soybean seeds, before oil extraction, with different concentrations of biotic elicitors such as chitosan and jasmonic acid also significantly reduced the levels of residual PLs as well as the TBA number. The biotic elicitor treatment could thus prove to be an important strategy for the reduction of off-flavour in protein-rich soymeal.

Identifying or designing low phytic acid soybean demands an improved understanding of its dynamics and a need for a reliable and accurate phytate quantification method and hence an improved quantitative technique for accurate and rapid quantification of phytic acid (PA) using high-performance liquid chromatography is proposed in this paper. A rapid PA extraction method utilizing sonication of sample in 0.78 M HCl for 3 min followed by mechanical agitation and separation using strong anion exchange column in a vacuum manifold was optimized. The elution of PA was performed using a RP-C18 column with an isocratic mobile phase [Acetonitrile, 35 mM formic acid and tetrabutylammonium hydroxide (4.8: 5.1: 0.1, v/v/v)]. The modified method was rapid, accurate, precise, and reproducible with relative standard deviation of 1.80 and 3.01 % (n = 10, for 1 mg ml−1) for within and between days respectively with linearity (R2 = 0.999, P

Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l-1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l-1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T1 plants. Southern analysis of T1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

The coding sequence of inositol polyphosphate 6-/3-/5-kinase (GmIPK2) gene was identified and cloned from popular Indian soybean cultivar Pusa-16. The clone was predicted to encode 279 amino acids long, 30.97 kDa protein. Multiple sequence alignment revealed an inositol phosphate-binding motif, PxxxDxKxG throughout the IPK2 sequences along with other motifs unique to inositol phosphate kinase superfamily. Eight α-helices and eight β-strands in antiparallel β-sheets arrangement were predicted in the secondary structure of GmIPK2. The temporal analysis of GmIPK2 revealed maximum expression in the seed tissues during later stages of development while spatially the transcript levels were lowest in leaf and stem tissues. Endosperm-specific cis-regulatory motifs (GCN4 and Skn_1) which support high levels of expression, as observed in the developing seeds, were detected in its promoter region. The protein structure of GmIPK2 was modeled based on the crystal structure of inositol polyphosphate multikinase from Arabidopsis thaliana (PDB:4FRF) and subsequently docked with inositol phosphate ligands (PDB: 5GUG-I3P and PDB: 4A69-I0P). Molecular dynamics (MD) simulation established the structural stability of both, modeled enzyme and ligand-bound complexes. Docking in combination with trajectory analysis for 50 ns MD run confirmed the participation of Lys105, Lys126 and Arg153 residues in the formation of a network of hydrogen bonds to stabilize the ligand-receptor interaction. Results of the present study thus provide valuable information on structural and functional aspects of GmIPK2 which shall assist in strategizing our long-term goal of achieving phytic acid reduction in soybean by genetic modification of its biosynthetic pathway to develop a nutritionally enhanced crop in the future. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1343-7) contains supplementary material, which is available to authorized users.

γ-Tocopherol methyl transferase (γ-TMT) (EC 2.1.1.95) is the key enzyme of the tocopherol biosynthetic pathway that determines the α-tocopherol concentration in plants. The overexpression of γ-TMT has been a successful approach for α-tocopherol enrichment of most plants including soybean. The typical soybean varieties are rich in γ-tocopherol (constitutes nearly 65–70% of its total seed tocopherol pool), while α-tocopherol, the biologically most active form among all tocopherols, constitutes only 10% of the total tocopherol content. The identification of soybean varieties that have seed α-tocopherol as high as > 20% of the total tocopherols has shifted attention towards the breeding based approach for α-tocopherol enrichment of this crop. Previous research on this aspect suggests that polymorphisms in γ-TMT promoter might be associated with the high α-tocopherol concentration of some soybean varieties. To understand the molecular basis of genetic variation for α-tocopherol concentration in Indian varieties of soybean we cloned the 1.4 kb upstream promoter region of γ-TMT from a high α-tocopherol containing soybean variety (Bragg) as well as from a low α-tocopherol containing variety (DS 2706). Cloning of each of these promoters in pORE R2 vector having GUS reporter gene and the subsequent GUS assay revealed a slightly high promoter activity of Bragg γ-TMT as compared to DS 2706 γ-TMT. On promoter sequence analysis, no sequence polymorphisms were observed in the core promoter region of this gene. However, seven single nucleotide polymorphisms (SNPs) were observed outside the core promoter region. Further study based on deletion construct analysis of this promoter will elucidate the significance of these SNPs in influencing the activity of γ-TMT promoter and the α-tocopherol concentration.

Designing low-phytate crops without affecting the developmental process in plants had led to the identification of ABCC5 gene in soybean. The GmABCC5 gene was identified and a partial gene sequence was cloned from popular Indian soybean genotype Pusa16. Conserved domains and motifs unique to ABC transporters were identified in the 30 homologous sequences retrieved by BLASTP analysis. The homologs were analyzed for their evolutionary relationship and physiochemical properties. Conserved domains, transmembrane architecture and secondary structure of GmABCC5 were predicted with the aid of computational tools. Analysis identified 53 alpha helices and 31 beta strands, predicting 60% residues in alpha conformation. A three-dimensional (3D) model for GmABCC5 was developed based on 5twv.1.B (Homo sapiens) template homology to gain better insight into its molecular mechanism of transport and sequestration. Spatio-temporal real-time PCR analysis identified mid-to-late seed developmental stages as the time window for the maximum GmABCC5 gene expression, a potential target stage for phytate reduction. Results of this study provide valuable insights into the structural and functional characteristics of GmABCC5, which may be further utilized for the development of nutritionally enriched low-phytate soybean with improved mineral bioavailability.

Soybean genome encodes a family of four inositol 1,3,4 trisphosphate 5/6 kinases which belong to the ATP-GRASP group of proteins. Inositol 1,3,4 trisphosphate kinase-2 (GmItpk2), catalyzing the ATP-dependent phosphorylation of Inositol 1,3,4 trisphosphate (IP3) to Inositol 1,3,4,5 tetra phosphate or Inositol 1,3,4,6 tetra phosphate, is a key enzyme diverting the flux of inositol phosphate pool towards phytate biosynthesis. Although considerable research on characterizing genes involved in phytate biosynthesis is accomplished at genomic and transcript level, characterization of the proteins is yet to be explored. In the present study, we report the isolation and expression of single copy Itpk2 (948 bp) from Glycine max cv Pusa-16 predicted to encode 315 amino acid protein with an isoelectric point of 5.9. Sequence analysis revealed that GmITPK2 shared highest similarity (80%) with Phaseolus vulgaris. The predicted 3D model confirmed 12 α helices and 14 β barrel sheets with ATP-binding site close to β sheet present towards the C-terminus of the protein molecule. Spatio-temporal transcript profiling signified GmItpk2 to be seed specific, with higher transcript levels in the early stage of seed development. The present study using various molecular and bio-computational tools could, therefore, help in improving our understanding of this key enzyme and prove to be a potential target towards generating low phytate trait in nutritionally rich crop like soybean.

γ-Tocopherol methyl transferase (γ-TMT) (EC 2.1.1.95) is the key enzyme of the tocopherol biosynthetic pathway that determines the α-tocopherol concentration in plants. The overexpression of γ-TMT has been a successful approach for α-tocopherol enrichment of most plants including soybean. The typical soybean varieties are rich in γ-tocopherol (constitutes nearly 65–70% of its total seed tocopherol pool), while α-tocopherol, the biologically most active form among all tocopherols, constitutes only 10% of the total tocopherol content. The identification of soybean varieties that have seed α-tocopherol as high as > 20% of the total tocopherols has shifted attention towards the breeding based approach for α-tocopherol enrichment of this crop. Previous research on this aspect suggests that polymorphisms in γ-TMT promoter might be associated with the high α-tocopherol concentration of some soybean varieties. To understand the molecular basis of genetic variation for α-tocopherol concentration in Indian varieties of soybean we cloned the 1.4 kb upstream promoter region of γ-TMT from a high α-tocopherol containing soybean variety (Bragg) as well as from a low α-tocopherol containing variety (DS 2706). Cloning of each of these promoters in pORE R2 vector having GUS reporter gene and the subsequent GUS assay revealed a slightly high promoter activity of Bragg γ-TMT as compared to DS 2706 γ-TMT. On promoter sequence analysis, no sequence polymorphisms were observed in the core promoter region of this gene. However, seven single nucleotide polymorphisms (SNPs) were observed outside the core promoter region. Further study based on deletion construct analysis of this promoter will elucidate the significance of these SNPs in influencing the activity of γ-TMT promoter and the α-tocopherol concentration.

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.