Unravelling the effect of extraction on anthocyanin functionality and prebiotic potential.

Anthocyanins, considered as prebiotic ingredients for functional foods, were extracted from black soybean (BS), black grape (BG), black carrot (BCPm), and black rice (BR) using conventional solvent extraction (CSE) and microwave-assisted extraction (MAE). The study employed a split-plot design with CSE and MAE as main plot factors and anthocyanin extracts (AEs) as subplot factors. Anthocyanins were evaluated for stability (polymeric color, degradation index) and functionality (antioxidant capacity). Prebiotic potential on Lactobacillus rhamnosus, Lactobacillus acidophilus, Weissella confusa was assessed in fermented soymilk. MAE showed higher extraction yield than CSE in BG (3-fold), BS (2-fold), BCPm (1.2-fold), and BR (1.6-fold). Black grape (1255.76 mg/L) and black soybean (976.5 mg/L) had highest anthocyanin with better stability, functionality, and prebiotic potential. The SCFA concentration (propionic acid and butyric acid) increased significantly in BG fortified-fermented soymilk. Overall, anthocyanin-enriched soymilk exhibited higher prebiotic potential, with MAE as the superior extraction method for anthocyanin functionality and stability.

Current trends in the development of soy-based foods containing probiotics and paving the path for soy-synbiotics.

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 synergy, in which a prebiotic ingredient particularly favors the growth and activity of a probiotic micro-organism. The insights on emended bioactive profile, metabolic role, and potential health benefits of advanced soy-based probiotic and synbiotic hold a promise which can be profitably implemented to meet consumer needs. This article reviews the available knowledge about strategies to enhance the nutraceutical potential, mechanisms, and health-promoting effects of advanced soy-based probiotics. Traditional fermentation merged with diverse strategies to improve the efficiency and health benefits of probiotics considered vital, are also discussed.

Isoflavones Play a Potential Role in Off-Flavour Scavenging, with a Key Role of IFS2 in Isoflavone Accumulation in Soybean Seeds.

Research background: Soybean (Glycine max (L.) Merr) is a nutrient-rich crop with a high protein content and various bioactive compounds with health-promoting properties. Nevertheless, it is poorly accepted as a food by consumers due to its off-flavour. Due to the ubiquitous presence of isoflavones in soybeans, their inherent antioxidant potential and inhibitory effect on lipoxygenase activity, their sensory properties are currently being considered to mitigate the off-flavour. Experimental approach: In the present study, the content and composition of isoflavones in 17 soybean cultivars are determined. The correlation between the isoflavone mass fraction and lipid peroxidation was also established, using thiobarbituric acid (TBA) value and carbonyl compound concentration as indices for the development of off-flavour. Cloning, gene expression analysis and in silico analysis of isoflavone synthase isoforms (IFS1 and IFS2) were also performed. Results and conclusions: The total isoflavone mass fraction in soybean genotypes ranged from (153.5±7.2) µg/g for PUSA 40 to (1146±43) µg/g for Bragg. There was a moderately negative correlation between the indices of off-flavour formation and the genistein/daidzein ratio (p<0.1). However, the correlation with total isoflavone mass fraction was found to be insignificant, indicating complex interactions. Higher protein-protein interactions for the predicted structure of IFS2 with other biosynthesis enzymes and its comparatively higher expression in the Bragg than that of IFS1 indicated its more important role in isoflavone synthesis. Novelty and scientific contribution: The genistein/daidzein mass ratio was found to be an important factor in controlling off-flavour. IFS2 was identified as key to produce soybeans with high isoflavone mass fraction and potentially lower off-flavour formation.

Interactome of millet-based food matrices: A review.

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.

Thermal treatments reduce rancidity and modulate structural and digestive properties of starch in pearl millet flour.

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 molecular configuration and starch-sugar homeostasis: Key determinants of sweet sensory perception and starch hydrolysis in pearl millet (Pennisetum glaucum).

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.

Role of ATP-binding cassette transporters in maintaining plant homeostasis under abiotic and biotic stresses.

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'.

Expression profiling and in silico homology modeling of Inositol pentakisphosphate 2-kinase, a potential candidate gene for low phytate trait in soybean.

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 IP6. 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".

Study of subcellular localization of Glycine max γ-tocopherol methyl transferase isoforms in N. benthamiana.

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.

Analysis of γ-Tocopherol methyl transferase3 promoter activity and study of methylation patterns of the promoter and its gene body.

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.

Evaluation of enzyme and microwave-assisted conditions on extraction of anthocyanins and total phenolics from black soybean (Glycine max L.) seed coat.

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 ±â€¯0.01), Browning Index (BI) (0.202 ±â€¯0.02) and Degradation Index (DI) (0.140 ±â€¯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.

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

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.

Cytosine Methylation of Isoflavone Synthase Gene in the Genic Region Positively Regulates Its Expression and Isoflavone Biosynthesis in Soybean Seeds.

Plants, being sessile organisms, have evolved several dynamic mechanisms of gene regulation. Epigenetic modification especially cytosine methylation and demethylation actively regulates the expression of genes. To understand the role of cytosine methylation during isoflavonoid biosynthesis and accumulation, we performed cytosine methylation analysis in the coding region of two isoforms IFS1 and IFS2 gene, in two contrasting soybean genotypes differing in total isoflavone content (NRC37: high isoflavone; and NRC7: low isoflavone). The results indicated increased 5-mC in both the isoforms in NRC37 (∼20.51% in IFS2 and ∼85% in IFS1) compared with NRC7 (∼7.8% in IFS2 and ∼2.5% in IFS1) genotype, which signifies the positive role of 5-mC in the coding region of the gene leading to enhanced expression. In addition, temporal expression profiling [35 days after flowering (DAF), 45, 55, and 65 DAF] of both the isoforms showed increasing trend of accumulation in both the genotypes with maximum in NRC37 at 65 DAF. To further establish a correlation between methylation and expression of transcripts, we quantified the different isoforms of isoflavone in both the genotypes across all the stages. Therefore, the finding of this study would certainly increase our understanding of epigenetic regulation of isoflavone biosynthetic pathway mediated by the cytosine methylation that would assist molecular breeders to get high-performing soybean genotypes with better isoflavone yield.

Conserved miRNAs modulate the expression of potential transcription factors of isoflavonoid biosynthetic pathway in soybean seeds.

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.

Valorisation of black carrot pomace: microwave assisted extraction of bioactive phytoceuticals and antioxidant activity using Box-Behnken design.

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.

Molecular characterization, modeling, and docking analysis of late phytic acid biosynthesis pathway gene, inositol polyphosphate 6-/3-/5-kinase, a potential candidate for developing low phytate crops.

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 ß-strands in antiparallel ß-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.

Development and Evaluation of Low Phytic Acid Soybean by siRNA Triggered Seed Specific Silencing of Inositol Polyphosphate 6-/3-/5-Kinase Gene.

Soybean is one of the leading oilseed crop in the world and is showing a remarkable surge in its utilization in formulating animal feeds and supplements. Its dietary consumption, however, is incongruent with its existing industrial demand due to the presence of anti-nutritional factors in sufficiently large amounts. Phytic acid in particular raises concern as it causes a concomitant loss of indigestible complexed minerals and charged proteins in the waste and results in reduced mineral bioavailability in both livestock and humans. Reducing the seed phytate level thus seems indispensable to overcome the nutritional menace associated with soy grain consumption. In order to conceive our objective we designed and expressed a inositol polyphosphate 6-/3-/5-kinase gene-specific RNAi construct in the seeds of Pusa-16 soybean cultivar. We subsequently conducted a genotypic, phenotypic and biochemical analysis of the developed putative transgenic populations and found very low phytic acid levels, moderate accumulation of inorganic phosphate and elevated mineral content in some lines. These low phytic acid lines did not show any reduction in seedling emergence and displayed an overall good agronomic performance.

Molecular cloning and functional analysis of the promoter of γ-Tocopherol Methyl Transferase (γ-TMT) gene of soybean (Glycine max).

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.

Exploring the role of Inositol 1,3,4-trisphosphate 5/6 kinase-2 (GmITPK2) as a dehydration and salinity stress regulator in Glycine max (L.) Merr. through heterologous expression in E. coli.

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.

Characterization and molecular modeling of Inositol 1,3,4 tris phosphate 5/6 kinase-2 from Glycine max (L) Merr.: comprehending its evolutionary conservancy at functional level.

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 ß barrel sheets with ATP-binding site close to ß 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.