The nutrition and therapeutic potential of millets: an updated narrative review.

Millets are ancient small grains grown in arid and semiarid regions of the world. They are staple food for many people in Asia and Africa. They are abundant sources of minerals and vitamins, giving them the name Nutricereals. Moreover, millets contain valuable phytochemicals that impart therapeutic properties for various disorders and diseases, thus giving them nutraceutical value. A wide array of biochemical compounds are present in the plant parts as well as the grains. In the oldest texts of medicine in India and China, millets are mentioned for use for their medicinal value. There has been expanding interest and emerging facts about millets and their therapeutic uses. Ample evidence shows that consumption of millets amounts to correction of life style and metabolic disorders. Therapeutic properties of millets can be viewed in two ways, supplementary nutrition through minerals and vitamins, and therapeutic value through the presence of phytochemicals and specialty compounds that include flavonoids, phenolics, anthocyanidins and others that have antioxidant potential. Millets are gluten free, have low glycemic index and the phytochemicals aid in correction of lifestyle disorders and prevention of ailments like carcinogenesis. Supplementary benefits include treatment of anemia and calcium deficiency especially for pregnant women and young children. With the improvements in analytical methods for detection of various compounds, it is possible to identify the compound-specific genotypes in millets that can cater to the pharmacy industry. End-use specific genotypes can be bred to meet the demand. Millets being climate resilient, can contribute to a healthier life and better world through economic usage of natural resources.

A novel method for detecting conformation-dependent ß-glucans-single, double, and triple-helical from Shiitake mushroom.

This pioneering study explores the structural intricacies of therapeutic ß-glucan in Shiitake (Lentinula edodes), i.e. Lentinan (LNT). Lentinan, a neutral polysaccharide [ß-(1,3; 1,6) glucan], exists in three forms; single, double, and triple-helical, but conformation-dependent bioactivity studies are lacking. In this context, we meticulously assessed indigenous Shiitake accessions from Northeast India, unveiling the conformational spectrum of LNT through an innovative pipeline. The experiment approached the simultaneous estimation of total glucan (TG), triple helical glucan (THG), and single-double helical glucan (SDG). Profiling revealed the exceptional LNT content in DMRO-623 (TG: 46.74%, SDG: 9.34%, THG: 37.39%) which emerged as the highest documented to date. Beyond the culinary delight, this research and the novel approach to LNT quantification will create a pivotal platform for advanced mushroom research, offering prospects for novel discoveries, innovative applications, and therapeutic potential.

A novel micronutrients and methyl jasmonate cocktail of elicitors via seed priming improves drought tolerance by mitigating oxidative stress in rice (Oryza sativa L.).

Drought is a major limiting factor for rice (Oryza sativa L.) production globally, and a cost-effective seed priming technique using bio-elicitors has been found to have stress mitigating effects. Till date, mostly phytohormones have been preferred as bio-elicitors, but the present study is a novel attempt to demonstrate the favorable role of micronutrients-phytohormone cocktail, i.e., iron (Fe), zinc (Zn), and methyl jasmonate (MJ) via seed priming method in mitigating the deleterious impacts of drought stress through physio-biochemical and molecular manifestations. The effect of cocktail/priming was studied on the relative water content, chlorophyll a/b and carotenoid contents, proline content, abscisic acid (ABA) content, and on the activities of ascorbate peroxidase (APX), superoxide dismutase (SOD), NADPH oxidase (Nox), and catalase (CAT). The expressions of drought-responsive genes OsZn-SOD, OsFe-SOD, and Nox1 were found to be modulated under drought stress in contrasting rice genotypes -N-22 (Nagina-22, drought-tolerant) and PS-5 (Pusa Sugandh-5, drought-sensitive). A progressive rise in carotenoids (10-19%), ABA (18-50%), proline (60-80%), activities of SOD (27-62%), APX (46-61%), CAT (50-80%), Nox (16-30%), and upregulated (0.9-1.6-fold) expressions of OsZn-SOD, OsFe-SOD, and Nox1 genes were found in the primed plants under drought condition. This cocktail would serve as a potential supplement in modern agricultural practices utilizing seed priming technique to mitigate drought stress-induced oxidative burst in food crops.

Hydrogen Peroxide and GA3 Levels Regulate the High Night Temperature Response in Pistils of Wheat (Triticum aestivum L.).

High night temperature (HNT) impairs crop productivity through the reproductive failure of gametes (pollen and pistil). Though female gametophyte (pistil) is an equal partner in the seed-set, the knowledge of the antioxidant system(s) and hormonal control of HNT tolerance or susceptibility of pistils is limited and lacking. The objectives of this study were to determine the antioxidant mechanism for homeostatic control of free radicals, and the involvement of abscisic acid (ABA) and gibberellic acid (GA3) in HNT stress protection in the wheat pistils of contrasting wheat genotypes. We hypothesized that HNT tolerance is attributed to the homeostatic control of reactive oxygen species (ROS) and hormonal readjustment in pistils of the tolerant genotype. The ears of two contrasting wheat genotypes-HD 2329 (susceptible) and Raj 3765 (tolerant) were subjected to two HNTs (+5 °C and +8 °C) over ambient, in the absence and presence of dimethylthiourea (DMTU), a chemical trap of hydrogen peroxide (H2O2). Results showed that HNTs significantly increased ROS in pistils of susceptible genotype HD 2329 to a relatively greater extent compared to tolerant genotype Raj 3765. The response was similar in the presence or absence of DMTU, but the H2O2 values were lower in the presence of DMTU. The ROS levels were balanced by increased activity of peroxidase under HNT to a greater extent in the tolerant genotype. Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) activity was inversely related to H2O2 production within a critical range in Raj 3765, indicating its modulation by H2O2 levels as no change was observed at the transcriptional level. The hormonal status showed increased ABA and decreased GA3 contents with increasing temperature. Our study elucidates the role of H2O2 and GA3 in stress tolerance of pistils of tolerant genotype where GAPC acts as a ROS sensor due to H2O2-mediated decrease in its activity.

Recent advances in oral delivery of bioactive molecules: Focus on prebiotic carbohydrates as vehicle matrices.

Controlled oral delivery of bioactive molecules remains a promising platform for the food and biomedical realm. Nonetheless, there are many bottlenecks to the efficient oral bioactive delivery that necessitates the development of advanced approaches. In recent years, prebiotic carbohydrates have drawn surging interest for targeted bioactive delivery due to their potential of multi-stimuli release mechanisms. Harnessing prebiotic-based vehicles confers novel possibilities for intact oral bioactive delivery, improving their bioavailability and efficacy. This critical review updates state of the art on progresses in oral delivery of natural active agents via prebiotic carbohydrates. We offer the latest advances concerning prebiotic-based vehicles (i.e., pH/time-dependent systems, enzyme-sensitive polymers, and colonic microbiota-dependent vehicles), emphasizing their key attributes to attaining controlled/targeted bioactive delivery to the intended locus. Finally, we discuss safety considerations, challenges, and future perspectives toward advances in the field.

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.

Starch-lipid interaction alters the molecular structure and ultimate starch bioavailability: A comprehensive review.

Starch bioavailability which results in eliciting postprandial glycaemic response, is a trait of great significance and is majorly influenced by the physical interaction among the matrix components governed by their molecular structure as well as dynamics. Among physical interactions limiting starch bioavailability, starch and any guest molecules like lipid interact together to alter the molecular structure into a compact V-type arrangement endorsing the processed crystallinity, thus limiting carbolytic enzymatic digestion and further bioavailability. Considering the importance of starch-lipid dynamics affecting bioavailability, intensive research based on endogenous (internal lipids which are embedded into the food matrix) as well as exogenous (those are added from outside into the food matrix during processing like cooking) lipids have been carried out, endorsing physical interactions at colloidal and microstructural levels. The shared insights on such binary (starch-lipid) interactions revealed the evolution of characterization techniques as well as their role on altering the functional and nutritional value. It is very much vital to have a thorough understanding about the mechanisms on the molecular level to make use of these matrix interactions in the most efficient way, while certain basic questions are still remaining unaddressed. Do starch - lipid complexation affects the ultimate starch bioavailability? If so, then whether such complexation ability depends on amylose - fatty acid/lipid content? Whether the complexation is influenced further by fatty acid type/concentration/chain length or saturation? Further comprehending this, whether the altered bioavailability by binary (starch-lipid) could further be affected by ternary (starch-lipid-protein) and quaternary (starch-lipid-protein-phenolics) interactions are also discussed in this comprehensive review.

Role of nutraceutical starch and proanthocyanidins of pigmented rice in regulating hyperglycemia: Enzyme inhibition, enhanced glucose uptake and hepatic glucose homeostasis using in vitro model.

Dysregulation of glucose homeostasis result in hyperglycemia and pigmented rice, unique combination of high quality starch and phenolics has the potential in regulating it. In this study, pigmented rice was characterized in terms of nutraceutical starch (NS) and phenolic content. Further the effect of rice phenolics on carbolytic enzyme inhibition, glucose uptake, hepatic glucose homeostasis and anti-glycation ability was analyzed in vitro. The most relevant effect on enzyme inhibition (α-amylase: IC50-42.34 µg/mL; α-glucosidase: IC50:63.89 µg/mL), basal uptake of glucose (>39.5%) and anti-glycation ability (92%) was found in red rice (RR), than black rice (BR). The role of RR phenolics in regulating glucose homeostasis was deciphered using hepatic cell line system, which found up-regulation of glucose transporter 2 (GLUT2) and glycogen synthase 2 (GYS2); while expression of gluconeogenic genes were found down regulated. To our knowledge this study is the first report validating the role of starch-phenolic quality towards anti-hyperglycemic effect of RR.

Cooking fat types alter the inherent glycaemic response of niche rice varieties through resistant starch (RS) formation.

Studies over the decades highlighted the role of lipids in modulating inherent glycaemic response of rice, still much needed to elucidate how the chain length and saturation of fatty acid (FA) influence this. Hence in this study, we investigated the in vitro glycaemic response, starch-lipid complexing ability and resistant starch (RS) formation in three rice types [white rice (WR), black rice (BR) and red rice (RR)] cooked with four fats [ghee, coconut oil (CO), virgin coconut oil (VCO) and rice bran oil (RBO)], with three cooking conditions ('before', 'during' and 'after'). Inherent glycaemic response was found least in RR (81.9%) and among the fats used, RBO rich in long chain unsaturated FA (72.6%) further reduced the least glycaemic response with maximum complexing ability and enriched RS content. Cooking conditions also resulted significant variation in the parameters studied, the most significant effect with complexing ability (28.67%) and RS (2.26%) observed when RBO added 'during' with RR. FTIR fingerprint within 950 to 1200 cm-1 region validated the complex interactions of amylose among FA, alcohols and acids present in the RBO. This is the first report proposing a 'lipid induced resistance towards glycaemic response' model highlighting the importance of FA type towards modulating the molecular configuration, complexing ability and RS-V formation.

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".

Pullulanase activity: A novel indicator of inherent resistant starch in rice (Oryza sativa. L).

Starch quality studies over the decades highlighted the immense health benefits of resistant starch (RS), but still much is needed to elucidate the science behind its inherent formation. Till date, there is no report to establish the relationship between inherent RS content and pullulanase (PUL) activity in any of the crops. In this study, we emphasize the novel role of debranching enzyme, PUL towards inherent RS formation, using rice as a model crop. Biochemical analysis of 51 rice genotypes for amylose content (AC) revealed a good amount of variation ranging from 7.03 to 37.33%. Further, hierarchical clustering which resulted in 11 genotypes of varying RS (0.33-2.7%), highlighted medium dependency towards amylose and low dependency towards amylopectin content. The discrete differences in microstructure, unimodal distribution and tight packing of starch granules observed in higher RS genotype indicated the higher possibility of compact cluster structure of amylopectin, modulated by PUL. Qualitative and quantitative assays performed validated the relevant role of PUL towards inherent RS content with very high dependency score (R2 = 0.98). This is the first report regarding the fact that higher PUL activity contribute to inherent RS using novel hypothetical 'Pullulanase-Amylopectin Trimming Model'.

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.

Starch accumulation in rice grains subjected to drought during grain filling stage.

Drought stress during the grain filling stage severely affects the quality and quantity of starch in rice grains. The enzymes such as ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) and starch synthase (SS, EC 2.4.1.21) are the key regulatory enzymes involved in the starch biosynthesis. In this study, the activity of the AGPase and starch synthase (SS) was correlated with the qualitative and quantitative parameters such as sucrose, starch, amylose, amylopectin, and resistant starch in leaves, roots, and grains of drought tolerant (N22) and drought susceptible (IR64) cultivars under applied water deficit stress (WDS). Drought stress enhanced the remobilization of stored starch from leaves to developing rice grains which was positively correlated with a decrease in the starch and starch synthase activity in leaves. Starch accumulation in developing grains was positively correlated with an increase in the AGPase and SS activity under drought. It was found that starch, amylopectin, and sucrose content in developing grains increased under water deficit stress (WDS), while amylose content decreased in both the varieties. However, in leaves, the SS activity decreased while AGPase activity was found to be increased under WDS in both varieties. Decreased starch content in matured grains was due to shortening of grain filling stage as drought stress caused early plant senescence. Yield reduction under drought was more in susceptible variety IR64 as compared to tolerant genotype N22.

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.

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.

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.

Molecular modeling and in silico characterization of GmABCC5: a phytate transporter and potential target for low-phytate crops.

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.