Assessment of health risk, genotoxicity, and thiol compounds in Trigonella foenum-graecum (Fenugreek) under arsenic stress.

Arsenic (As) traces have been reported worldwide in vegetables and crops cultivated in As-polluted soils. Being carcinogenic, the presence of As in edibles is of great concern as it ultimately reaches humans and animals through the food chain. Besides, As toxicity adversely affects the growth, physiology, metabolism, and productivity of crops. In the present study, Trigonella foenum-graecum (Fenugreek) was exposed to the As stress (0, 50, 100, and 150 µM sodium arsenate) for a week. Further, evaluation of As accumulation in roots and shoots, magnitude and visualization of oxyradicals, and thiol-based defence offered by Fenugreek was assessed. The root and leaf accumulated 258-453 µg g-1 dry wt (DW) and 81.4-102.1 µg g-1 DW of As, respectively. An arsenic-mediated decline in the growth index and increase in oxidative stress was noted. Arsenic stress modulated the content of thiol compounds; especially cysteine content increased from 0.36 to 0.43 µmole g-1 FW protein was noted. Random Amplified Polymorphic DNA (RAPD)-based analysis showed DNA damage in As-treated plants. Health risk assessment parameters showed that As concentration in the consumable plant shoot was below the critical hazard level (hazard quotient < 1). Moreover, T. foenum-graecum showed varied responses to As-induced oxidative stress with applied concentrations (150 µM being more toxic than lower concentrations). In addition, the RAPD profile and level of thiol compounds were proved significant biomarkers to assess the As toxicity in plants. The conclusion of this study will help users of fenugreek to have a clue and create awareness regarding the consumption.

In Silico Analysis of PTP1B Inhibitors and TLC-MS Bioautography-Based Identification of Free Radical Scavenging and α-Amylase Inhibitory Compounds from Heartwood Extract of Pterocarpus marsupium.

Type 2 diabetes mellitus leads to metabolic impairment caused by insulin resistance and hyperglycemia, giving rise to chronic diabetic complications and poor disease prognosis. The heartwood of Pterocarpus marsupium has been used in Ayurveda for a long time, and we sought to find the actual mechanism(s) driving its antidiabetic potential. Methanol was used to prepare the extract using a Soxhlet extraction, and the identification of metabolites was performed by thin-layer chromatography (TLC) and ultraperformance-liquid chromatography and mass spectroscopy (UP-LCMS). The antioxidant potential of methanolic heartwood extract of Pterocarpus marsupium MHPM was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and a reducing power assay. The α-amylase and α-glucosidase enzyme inhibitory potential of MHPM were investigated for their antidiabetic activity against acarbose. TLC-MS-bioautography was performed to identify the compounds responsible for possible antioxidant and antidiabetic activities. Moreover, targeting protein tyrosine phosphatase 1B (PTP1B), a key regulator of insulin resistance, by identified metabolites from MHPM through molecular docking and all-atom molecular dynamics (MD) simulations was also undertaken, suggesting its potential as an antidiabetic herb. The IC50 of free-radical scavenging activity of MHPM against DPPH was 156.342 ± 10.70 µg/mL. Further, the IC50 values of MHPM in α-amylase and α-glucosidase enzymatic inhibitions were 158.663 ± 10.986 µg/mL and 180.21 ± 11.35 µg/mL, respectively. TLC-MS-bioautography identified four free radical scavenging metabolites, and vanillic acid identified by MS analysis showed both free radical scavenging activity and α-amylase inhibitory activity. Among the identified metabolites from MHPM, epicatechin showed significant PTP1B docking interactions, and its MD simulations revealed that PTP1B forms a stable protein-ligand complex with epicatechin throughout the progression, which indicates that epicatechin may be used as a promising scaffold in the development of the antidiabetic drug after isolation from Pterocarpus marsupium. Overall, these findings imply that Pterocarpus marsupium is a source of valuable metabolites that are accountable for its antioxidant and antidiabetic properties.

Heartwood Extract of Pterocarpus marsupium Roxb. Offers Defense against Oxyradicals and Improves Glucose Uptake in HepG2 Cells.

Diabetes mellitus leads to cellular damage and causes apoptosis by oxidative stress. Heartwood extract of Pterocarpus marsupium has been used in Ayurveda to treat various diseases such as leprosy, diabetes, asthma, and bronchitis. In this study, we worked out the mechanism of the antidiabetic potential of methanolic heartwood extract of Pterocarpus marsupium (MPME). First, metabolic profiling of MPME was done using gas chromatography-mass spectrometry (GCMS), ultra-performance liquid chromatography-mass spectroscopy (UPLC-MS), and high-performance thin-layer chromatography (HPTLC) to identify phenols, flavonoids, and terpenoids in MPME. Biological studies were carried out in vitro using the HepG2 cell line. Many antidiabetic compounds were identified including Quercetin. Methanolic extract of MPME (23.43 µg/mL-93.75 µg/mL) was found to be safe and effective in reducing oxyradicals in HepG2 cells. A concentration of 93.75 µg/mL improved glucose uptake efficiently. A significant decrease in oxidative stress, cell damage, and apoptosis was found in MPME-treated HepG2 cells. The study suggests that the heartwood of Pterocarpus marsupium offers good defense in HepG2 cells against oxidative stress and improves glucose uptake. The results show the significant antidiabetic potential of MPME using a HepG2 cell model. The effect seems to occur by reducing oxidative stress and sensitizing the cells towards glucose uptake, hence lowering systemic glucose levels, as well as rescuing ROS generation.

Impact of Ferrous Sulfate on Thylakoidal Multiprotein Complexes, Metabolism and Defence of Brassica juncea L. under Arsenic Stress.

Forty-day-old Brassica juncea (var. Pusa Jai Kisan) plants were exposed to arsenic (As, 250 µM Na2HAsO4·7H2O) stress. The ameliorative role of ferrous sulfate (2 mM, FeSO4·7H2O, herein FeSO4) was evaluated at 7 days after treatment (7 DAT) and 14 DAT. Whereas, As induced high magnitude oxidative stress, FeSO4 limited it. In general, As decreased the growth and photosynthetic parameters less when in the presence of FeSO4. Furthermore, components of the antioxidant system operated in better coordination with FeSO4. Contents of non-protein thiols and phytochelatins were higher with the supply of FeSO4. Blue-Native polyacrylamide gel electrophoresis revealed an As-induced decrease in almost every multi-protein-pigment complex (MPC), and an increase in PSII subcomplex, LHCII monomers and free proteins. FeSO4 supplication helped in the retention of a better stoichiometry of light-harvesting complexes and stabilized every MPC, including supra-molecular complexes, PSI/PSII core dimer/ATP Synthase, Cytochrome b6/f dimer and LHCII dimer. FeSO4 strengthened the plant defence, perhaps by channelizing iron (Fe) and sulfur (S) to biosynthetic and anabolic pathways. Such metabolism could improve levels of antioxidant enzymes, and the contents of glutathione, and phytochelatins. Important key support might be extended to the chloroplast through better supply of Fe-S clusters. Therefore, our results suggest the importance of both iron and sulfur to combat As-induced stress in the Indian mustard plant at biochemical and molecular levels through enhanced antioxidant potential and proteomic adjustments in the photosynthetic apparatus.

From design to validation of CRISPR/gRNA primers towards genome editing.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. There are many online platforms/software for the design of gRNAs and related primers. However, there are concerns in design regarding off-site deletions besides knocking out sequences in the target genes. Nonetheless, a well known robust platform for CRISPR/gRNA primers design is CRISPRdirect. We demonstrate the use of this tool in the design of CRISPR/gRNA primers for soluble starch synthases (SSS) II-1, 2, and 3 genes in the Oryza sativa genome followed by the PCR-mediated amplification of SSS genes with corresponding confirmation towards genome editing having improved phenotype features.

Differential impact of some metal(loid)s on oxidative stress, antioxidant system, sulfur compounds, and protein profile of Indian mustard (Brassica juncea L.).

Levels of arsenic (As), chromium (Cr), and copper (Cu) are increasing in the soils worldwide. Such contaminants cause toxicity in the plant systems which adversely affects growth and productivity. The objective of the present investigation was to elucidate individual and combined effects of As, Cr, and Cu (100 µM each) stress in metal hyper-accumulator plant Indian mustard (Brassica juncea L.), exposed for a week. The highest accumulation was in the roots and in decreasing order viz. Cu > As > Cr. The magnitude of oxidative stress was maximal in combined stress, followed by As, Cr, and then Cu stress. Glutathione in conjunction with glutathione reductase, glutathione peroxidase, and glutathione S-transferase increased in all set of stress treatments, notably when exposed to Cr alone. In addition, the level of sulfur-rich compounds like cysteine, phytochelatins, and non-protein thiols increased under each stress indicating efficient coupling of the enzyme system and sulfur-containing compounds during stress conditions. The highest tolerance or growth index of plants was recorded for Cu. Protein profiling of leaf tissues showed modulation of protein patterns in each stress. Mediator of RNA polymerase II transcription subunit 1 isoform X1, RuBisCO (large subunit), and ribosomal protein S3 proteins were more abundant under Cr and Cu stress. Zinc finger A20/AN1 domain-containing stress-associated protein 5-like protein was more abundant under Cu stress. HSP (15.7 kDa) and autophagy protein 5-like were in higher abundance under As and combined stress. Our results suggest that Indian mustard has a differential mode of defense against a particular stressor at the level of protein expression profile.

Drought mediated physiological and molecular changes in muskmelon (Cucumis melo L.).

Water deficiency up to a certain level and duration leads to a stress condition called drought. It is a multi-dimensional stress causing alteration in the physiological, morphological, biochemical, and molecular traits in plants resulting in improper plant growth and development. Drought is one of the major abiotic stresses responsible for loss of crops including muskmelon (Cucumis melo. L). Muskmelon genotype SC-15, which exhibits high drought resistance as reported in our earlier reports, was exposed to deficient water condition and studied for alteration in physiological, molecular and proteomic profile changes in the leaves. Drought stress results in reduced net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration (E) rate. With expanded severity of drought, declination recorded in content of total chlorophyll and carotenoid while enhancement observed in phenol content indicating generation of oxidative stress. In contrary, activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol (POD) were increased under drought stress. Peptide mass fingerprinting (PMF) showed that drought increased the relative abundance of 38 spots while decreases10 spots of protein. The identified proteins belong to protein synthesis, photosynthesis, nucleotide biosynthesis, stress response, transcription regulation, metabolism, energy and DNA binding. A drought-induced MADS-box transcription factor was identified. The present findings indicate that under drought muskmelon elevates the abundance of defense proteins and suppresses catabolic proteins. The data obtained exhibits possible mechanisms adopted by muskmelon to counter the impacts of drought induced stress.

Proteomic and ecophysiological responses of soybean (Glycine max L.) root nodules to Pb and hg stress.

BACKGROUND: Lead (Pb) and mercury (Hg) are persistent hazardous metals in industrially polluted soils which can be toxic in low quantities. Metal toxicity can cause changes at cellular and molecular level which should be studied for better understanding of tolerance mechanism in plants. Soybean (Glycine max L.) is an important oilseed crop of the world including India. Indian soils growing soybean are often contaminated by Pb and Hg. The aim of this study was to explore how soybean root nodule responds to Pb and Hg through proteomic and ecophysiological alterations in order to enhance tolerance to metal stress. RESULTS: Soybean plants were exposed to Pb (30 ppm PbCl2) and Hg (0.5 ppm HgCl2) to study histological, histochemical, biochemical and molecular response of N2-fixing symbiotic nodules. Both Pb and Hg treatment increased the level of oxidative stress in leaves and nodules. Chlorosis in leaves and morphological/anatomical changes in nodules were observed. Activities of ascorbate peroxidase, glutathione reductase and catalase were also modulated. Significant changes were observed in abundance of 76 proteins by Pb and Hg. Pb and Hg influenced abundance of 33 proteins (17 up and 16 down) and 43 proteins (33 up and 10 down), respectively. MS/MS ion search identified 55 proteins which were functionally associated with numerous cellular functions. Six crucial proteins namely catalase (CAT), allene oxide synthase (AOS), glutathione S-transferase (GST), calcineurin B like (CBL), calmodulin like (CML) and rapid alkalinisation factor (RAF) were selected for transcript abundance estimation. The qRT-PCR based real time expression exhibited a positive correlation with proteomics expression except for GST and RAF. CONCLUSION: Soybean root nodule responds to metal stress by increased abundance of defence, development and repair related proteins. An efficient proteomic modulation might lead to metal-induced stress tolerance in N2-fixing nodules. Although concentrations of Pb and Hg used in the study cannot be considered equimolar, yet Hg seems to induce more changes in nodule proteomic profile, and higher damage to both bacteroides and root anatomy.