Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata).

KEY MESSAGE: Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

A Lumenal Loop Associated with Catalytic Asymmetry in Plant Vacuolar H+-Translocating Pyrophosphatase.

Membrane-integral inorganic pyrophosphatases (mPPases) couple pyrophosphate hydrolysis with H+ and Na+ pumping in plants and microbes. mPPases are homodimeric transporters with two catalytic sites facing the cytoplasm and demonstrating highly different substrate-binding affinities and activities. The structural aspects of the functional asymmetry are still poorly understood because the structure of the physiologically relevant dimer form with only one active site occupied by the substrate is unknown. We addressed this issue by molecular dynamics (MD) simulations of the H+-transporting mPPase of Vigna radiata, starting from its crystal structure containing a close substrate analog (imidodiphosphate, IDP) in both active sites. The MD simulations revealed pre-existing subunit asymmetry, which increased upon IDP binding to one subunit and persisted in the fully occupied dimer. The most significant asymmetrical change caused by IDP binding is a 'rigid body'-like displacement of the lumenal loop connecting α-helices 2 and 3 in the partner subunit and opening its exit channel for water. This highly conserved 14-19-residue loop is found only in plant vacuolar mPPases and may have a regulatory function, such as pH sensing in the vacuole. Our data define the structural link between the loop and active sites and are consistent with the published structural and functional data.

Effect of storage states on stability of three organophosphorus insecticide residues on cowpea samples.

BACKGROUND: The stability of pesticide residues in stored samples is very important to ensure the quality of data about the residues. The evaluation of pesticide residues in food and environment samples is an important means to ensure food quality and protect consumers against potential dietary risks. Improper storage of pesticide residue samples may result in loss of pesticide and unreliable data, which could affect safety assessments. RESULTS: The influences of storage conditions, including temperature (-20 °C, 4 °C, and ambient temperature) and sample state (homogenized state and coarsely chopped state) on the storage stability of dichlorvos, malathion, and diazinon on cowpea were studied. Dichlorvos and malathion were more stable in an homogenized state than in a coarsely chopped state. At 4 °C, the residual dichlorvos in the coarsely chopped state and the homogenized state, respectively, was 12% and 69%; the residual malathion was 26% and 92%, respectively. Dichlorvos suffered a large loss of 89% and 59% for coarsely chopped and homogenized cowpea, even at -20 °C. It was obvious that the stability of dichlorvos and malathion were more affected by storage state than diazinon. The stability of diazinon was significantly affected by temperature. The effect of storage state and temperature on stability is likely to be correlated with enzymes in the matrix, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). CONCLUSION: The optimal stable storage conditions for three organophosphorus insecticides residues on cowpea were in the homogenized state and under a lower temperature. © 2021 Society of Chemical Industry.

Functional Characterization of Mung Bean Meal Protein-Derived Antioxidant Peptides.

The aim of this work was to characterize the antioxidant properties of some of the peptides present in bromelain mung bean meal protein hydrolysate (MMPH). The MMPH was subjected to two rounds of bioassay-guided reversed-phase HPLC separation followed by peptide identification in the most potent fractions using tandem mass spectrometry. Twelve antioxidant peptides, namely, HC, CGN, LAN, CTN, LAF, CSGD, MMGW, QFAAD, ERF, EYW, FLQL, and QFAW were identified and assayed for antioxidant properties. CTN, HC, CGN, and CSGD were the most potent (p < 0.05) DPPH radical scavengers with EC50 values of 0.30, 0.29, 0.28, and 0.30 mg/mL, respectively, which are lower than the 0.03 mg/mL obtained for reduced glutathione (GSH). CTN, HC, CGN, and CSGD exhibited the most potent (p < 0.05) scavenging activities against hydroxyl and superoxide radicals with EC50 values that are similar to those of GSH. The cysteine-containing peptides also had stronger ferric reducing antioxidant power and metal chelation activity than peptides devoid of cysteine. In contrast, MMGW, ERF, and EYW had poor radical scavenging and metal chelation activities. We conclude that the availability of the sulfhydryl group may have enhanced antioxidant potency while the presence of bulky groups such phenylalanine and tryptophan had an opposite effect.

Cloning, purification and characterisation of cytosolic fructose-1,6-bisphosphatase from mung bean (Vigna radiata).

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01-10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 µM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (-2.58 kcal·mol-1) and molecular distance (4.2 Å). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.

Atomic structures of respiratory complex III2, complex IV, and supercomplex III2-IV from vascular plants.

Mitochondrial complex III (CIII2) and complex IV (CIV), which can associate into a higher-order supercomplex (SC III2+IV), play key roles in respiration. However, structures of these plant complexes remain unknown. We present atomic models of CIII2, CIV, and SC III2+IV from Vigna radiata determined by single-particle cryoEM. The structures reveal plant-specific differences in the MPP domain of CIII2 and define the subunit composition of CIV. Conformational heterogeneity analysis of CIII2 revealed long-range, coordinated movements across the complex, as well as the motion of CIII2's iron-sulfur head domain. The CIV structure suggests that, in plants, proton translocation does not occur via the H channel. The supercomplex interface differs significantly from that in yeast and bacteria in its interacting subunits, angle of approach and limited interactions in the mitochondrial matrix. These structures challenge long-standing assumptions about the plant complexes and generate new mechanistic hypotheses.

Most living things including plants and animals use respiration to release energy from food. Respiration requires the activity of five large protein complexes typically called complex I, II, III, IV and V. Sometimes these complexes combine to form supercomplexes. The complexes are similar across plants, animals and other living things, but there are also many differences. Detailed structures of the respiratory complexes have been determined for many species of animals, fungi and bacteria, highlighting similarities and differences between organisms, and providing clues as to how respiration works. Yet, there is still a lot to learn about these complexes in plants. To bridge this gap, Maldonado et al. used a technique called cryo electron microscopy to study the structure of complexes III and IV and the supercomplex they form in the mung bean. This is the first study of the detailed structure of these two complexes in plants. The results showed many similarities to other species, as well as several features that are specific to plants. The way the two complexes interact to form a supercomplex is different than in other species, as are several other, smaller, structural features. Further examination of complex III revealed that it is flexible and that movements are coordinated across the length of the complex. Maldonado et al. speculate that this may allow it to coordinate its role in respiration with its other cellular roles. Understanding how plant respiratory complexes work could lead to improvements in crop yields or, since respiration is required for survival, result in the development of herbicides that block respiration in plants more effectively and specifically. Further researching the structure of the plant respiratory complexes and supercomplexes could also shed light on how plants adapt to different environments, including how they change to survive global warming.

Soluble and Cross-Linked Aggregated Forms of α-Galactosidase from Vigna mungo Immobilized on Magnetic Nanocomposites: Improved Stability and Reusability.

α-Galactosidases hold immense potential due to their biotechnological applications in various industrial and functional food sectors. In the present study, soluble and covalently cross-linked aggregated forms of a low molecular weight, thermo-labile α-galactosidase from Vigna mungo (VM-αGal) seeds were immobilized onto chitosan-coated magnetic nanoparticles for improved stability and repeated usage by magnetic separation. Parameters like precipitants (type, amount, and ratio), glutaraldehyde concentration, and enzyme load were optimized for the preparation of chitosan-coated magnetic nanocomposites of cross-linked VM-αGal (VM-αGal-MC) and VM-αGal (VM-αGal-M) resulted in 100% immobilization efficiency. Size and morphology of VM-αGal-M were studied through dynamic light scattering (DLS) and scanning electron microscopy (SEM), while Fourier transform infrared spectroscopy (FTIR) was used to study the chemical composition of VM-αGal-MC and VM-αGal-M. VM-αGal-MC and VM-αGal-M were found more active in a broad range of pH (3-8) and displayed optimal temperatures up to 25 °C higher than VM-αGal. Addition of non-ionic detergents (except Tween-40) improved VM-αGal-MC activity by up to 44% but negatively affected VM-αGal-M activity. Both VM-αGal-MC (15% residual activity after 21 min at 85 °C, Ed 92.42 kcal/mol) and VM-αGal-M (69.0% residual activity after 10 min at 75 °C, Ed 39.87 kcal/mol) showed remarkable thermal stability and repeatedly hydrolyzed the substrate for 10 cycles.

Phospholipase D inhibitors screening: Probing and evaluation of ancient and novel molecules.

Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.

Atomic structure of a mitochondrial complex I intermediate from vascular plants.

Respiration, an essential metabolic process, provides cells with chemical energy. In eukaryotes, respiration occurs via the mitochondrial electron transport chain (mETC) composed of several large membrane-protein complexes. Complex I (CI) is the main entry point for electrons into the mETC. For plants, limited availability of mitochondrial material has curbed detailed biochemical and structural studies of their mETC. Here, we present the cryoEM structure of the known CI assembly intermediate CI* from Vigna radiata at 3.9 Šresolution. CI* contains CI's NADH-binding and CoQ-binding modules, the proximal-pumping module and the plant-specific γ-carbonic-anhydrase domain (γCA). Our structure reveals significant differences in core and accessory subunits of the plant complex compared to yeast, mammals and bacteria, as well as the details of the γCA domain subunit composition and membrane anchoring. The structure sheds light on differences in CI assembly across lineages and suggests potential physiological roles for CI* beyond assembly.

Respiration is the process used by all forms of life to turn organic matter from food into energy that cells can use to live and grow. The final stage of this process relies on an intricate chain of protein complexes which produce the molecule that cells use for energy. Complexes in the chain are made up of specific proteins that are carefully assembled, often into discrete modules or intermediate complexes, before coming together to form the full protein complex. Understanding how these complexes are assembled provides important insights into how respiration works. The precise three-dimensional structure of these complexes has been identified for bacteria, yeast and mammals. However, less is known about how these respiration complexes form in plants. For this reason, Maldonado et al. studied the structure of an intermediate complex that is only found in plants, called Cl*. This intermediate structure goes on to form complex I ­ the largest complex in the respiration chain. A technique called cryo-electron microscopy was used to obtain a structure of Cl* at a near-atomic level of detail. This structure revealed how the proteins that make up Cl* fit together, highlighting differences and similarities in how plants assemble complex I compared to bacteria, yeast and mammals. Maldonado et al. also studied the activity of Cl*, leading to the suggestion that this complex may be more than just a stepping stone towards building the full complex I and could have its own role in the cell. The structure of this complex provides new insights into the respiration mechanism of plants and could help scientists improve crop production. For instance, new compounds may be able to block respiration in pests, while leaving the crop unharmed; or genetic modifications could create plants that respire more efficiently in different environments.

Physicochemical properties and in vitro digestibility of mung-bean starches varying amylose contents under citric acid and hydrothermal treatments.

In this study, starches of three mung bean cultivars (Vigna radiata) having different amylose contents were isolated and modified using a combination of citric acid and heat-moisture treatment (CA-HMT) or a combination of citric acid and annealing treatment (CA-ANN). Physicochemical properties and in vitro digestibility of native and treated mung bean starches were investigated. Resistant starch (RS) contents of the high-amylose, medium-amylose and low-amylose starches treated with CA-ANN were 41.1%, 35.7% and 27.1%, respectively, being higher than those treated with CA-HMT or native starches in the same variety. The CA-ANN did not affect the morphology and crystallinity of the starches. However, the starches treated by the CA-HMT were partly gelatinized and had higher degree of relative crystallinity as compared to the native starches. The DP¯s, swelling power, and viscosity of the treated starches were significantly lower than those of the native starches. The high-amylose mung bean starch was greatly affected by the treatments rather than medium- or low-amylose mung bean starch.

Effect of Low-Dose Nano Titanium Dioxide Intervention on Cd Uptake and Stress Enzymes Activity in Cd-Stressed Cowpea [Vigna unguiculata (L.) Walp] Plants.

Cadmium contamination of agricultural soils is a serious problem due to its toxic effects on health and yield of crop plants. This study investigates the potential of low-dose nano-TiO2 as soil nanoremediation on Cd toxicity in cowpea plants. To achieve this goal, cowpea seeds were germinated on Cd-spiked soils at 10 mg/kg for 14 days and later augmented with 100 mg nTiO2/kg (nTiO2-50 nm and bTiO2-68 nm, respectively). The results showed that chlorophylls were not altered by nano-TiO2 intervention. Cadmium partitioning in roots and leaves was reduced by the applied nano-TiO2 but significantly higher than control. Ascorbate peroxidase and catalase activities in roots and leaves were promoted by nano-TiO2 intervention compared to control and sole Cd, respectively. However, magnitudes of activity of enzyme activities were higher in nTiO2 compared to bTiO2 treatments. The enhanced enzymes activity led to reduced malonaldehyde content in plant tissues. The study concludes that soil application of nano-TiO2 could be a green alternative to ameliorate soil Cd toxicity in cowpea plants.

Cross-linked enzyme lyophilisates (CLELs) of urease: A new method to immobilize ureases.

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.

Rhamnogalacturonan I galactosyltransferase: Detection of enzyme activity and its hyperactivation.

Rhamnogalacturonan I (RG-I), one of the pectic components of the plant cell wall, is composed of a backbone of repeating disaccharide units of rhamnose and galacturonic acid, and side chains, such as galactans, arabinans, and arabinogalactans. The activity of RG-I galactosyltransferase, which transfers galactosyl residues to rhamnosyl residues in the RG-I backbone, has not been detected until now. Here, we detected galactosyltransferase activity in azuki bean epicotyls using fluorogenic RG-I oligosaccharide acceptors. This enzyme prefers oligosaccharides with a degree of polymerization more than 9. The enzyme activity was detected in the Golgi apparatus, which is the site of pectin synthesis. In vitro hyperactivation of this enzyme was also observed. Moreover, enzyme activity was increased up to 40-fold in the presence of cationic surfactants or polyelectrolytes.

Nitric oxide induced modulations in adventitious root growth, lignin content and lignin synthesizing enzymes in the hypocotyls of Vigna radiata.

The present study evaluated the role of nitric oxide (NO) in mediating adventitious root (AR) growth, lignification and related enzymatic changes in the hypocotyls of Vigna radiata. To meet the objectives, the changes in AR growth, lignin content, and the activities of enzymes-peroxidases, polyphenol oxidases, and phenylalanine ammonia lyases- with NO donor and its scavenger were monitored. Hypocotyls were cultivated in aqueous solution supplemented with different concentrations of SNP (sodium nitroprusside, NO donor compound) and its scavenging compound (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; cPTIO). Specifically, at low concentrations, SNP induced AR growth, increased the total lignin content and altered the activities of related oxidoreductases- peroxidases, polyphenol oxidases and phenylalanine ammonia lyases- which are involved in lignin biosynthesis pathway. At higher concentrations, a decline in AR growth and lignification was noticed. We analysed the function of NO in AR formation by depleting the endogenous NO using scavenging compound cPTIO. Hypocotyls grown in a medium supplemented with scavenger cPTIO exhibited significant decline in AR growth and the activities of lignin synthesizing enzymes. Application of NO scavenger showed that stimulatory properties on root lignification may be owing to NO itself. In addition, changes in AR growth were significantly correlated with these modified biochemical activities. Our analysis revealed that NO supplementation induces prominent alterations in lignin level during AR formation and this might be due to an alteration in the activity of lignin biosynthetic enzymes, which further affected the polymerization of monolignols and AR growth.

Orchestration of Cu-Zn SOD and class III peroxidase with upstream interplay between NADPH oxidase and PM H+-ATPase mediates root growth in Vigna radiata (L.) Wilczek.

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2- scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2-, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.

Physico-chemical properties, antioxidant activities and angiotensin-I converting enzyme inhibitory of protein hydrolysates from Mung bean (Vigna radiate).

Mung bean Protein hydrolyses (MBPHs) have attracted a great deal of attention due to their variety of biological activities. In present study, MBPHs were fractionate according to the molecular mass into three fractions of MBPHs-I (<3 kDa), MBPHs-II (3-10 kDa) and MBPHs-III (>10 kDa). Their antioxidant activity and angiotensin-I converting enzyme (ACE) inhibitory of were investigated in vitro. Results showed that the alcalase-derived hydrolysate exhibited the highest degree of hydrolysis (DH) and trichloroacetic acid-nitrogen soluble index (TCA-NSI) versus those of other enzyme hydrolysates. MBPHs-I presented the best scavenge DPPH, hydroxyl radicals, superoxide radicals, Fe2+ chelating activities, and the best ACE inhibitory activity (IC50 = 4.66 µg/mL) than that of MBPHs and MBPHs-III. And MBPHs-I rich in hydrophobic and aromatic amino acids, and its secondary structure mainly contain α-helix, ß-sheet and irregular coiled. Results indicated that MBPHs-I has a great potential as natural functional materials for supplement.

Possible interaction of ROS, antioxidants and ABA to survive osmotic stress upon acclimation in Vigna radiata L. Wilczek seedlings.

Acclimation is a process of adjustment to gradual environmental change that enables plants to survive further stress by triggering some tolerance mechanism possibly involving ABA, ROS and oxidative metabolism. Here we have studied acclimation responses in terms of the performances with regard to physiological (growth and relative water content) and biochemical (chlorophyll, carotenoids, protein, malondialdehyde, sugar content) attributes, ABA production and stomatal sensitivity to exogenous ABA, extracellular ROS production and activation of antioxidant system. Our study reveals that repeated exposure to short-term mild water stress simulated by polyethylene glycol (PEG-6000) induces acclimation in mung bean (Vigna radiata L. Wilczek) seedlings. Acclimation induced tolerance was associated with reduced leaf size and enhanced root growth, accumulation of soluble sugar as osmoprotectant, maintenance of water potential, lessening of membrane damage as indicated by lower MDA content. Acclimated mung bean seedlings have shown greater degree of tolerance through increased production of and enhanced sensitivity to ABA (as reflected by faster stomatal closure), enhanced production of extracellular O2.- and H2O2 and the elevated activities of antioxidative enzymes to control the oxidative burst. Taken together, the results convey that acclimated seedlings minimize osmotic stress-induced damage through a possible network of ABA, ROS and antioxidants.

Kinetic study of enzymatic α-galactoside hydrolysis in cowpea seeds.

The endogenous alpha-galactosidase activity of cowpea seeds was characterized and modelled assuming Michaelian behavior. The aim is to use the resulting knowledge to optimize alpha-galactoside degradation during the soaking-cooking process. In this study, the alpha-galactosidase enzyme from Wankoun cowpea was extracted and its enzymatic activity was analyzed as a function of temperature, pH and the presence of some inhibitors. Enzymatic activity was optimal around 35 °C and a pH of 5.8. Activation and inactivation energy was evaluated at 50 ±â€¯3 and 103 ±â€¯9 kJ.mol-1, respectively. The strongest inhibitor was galactose with an inhibition constant KI of 0.28 ±â€¯0.03 mM. Incubation of the enzyme extract with alpha-galactosides revealed a 10-h lag phase in the early stages that could be due to low pH, the action of inhibitors including galactose and the biosynthesis of alpha-galactosides. After the lag phase, the degradation of each alpha-galactoside occurred without the appearance of any intermediary product. The degradation of alpha-galactosides was observed with a Km of 1.7 ±â€¯0.3 mM for raffinose; 3.6 ±â€¯0.6 mM for stachyose and 15.9 ±â€¯0.1 mM for verbascose. A long soaking step around 35 °C is suggested to maximize the alpha-galactosides enzymatic degradation.

Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies.

The production of pure enzymes in high quantities is a proven strategy to study the catalytic mechanism as well as the solving of structure at the atomic scale for therapeutic or industrial purposes. Phospholipase D (PLD, EC 3.1.4.4) is found in a wide majority of living organisms and has been shown to be involved in signal transduction, vesicle trafficking, and membrane metabolism processes. Located at the membrane-cytoplasm interface, plant PLDs are soluble but also bear an evident hydrophobic aspect making challenging its expression and its purification in large quantity. So far there is no high-resolution three-dimensional structure for a eukaryotic PLD. The protocols herein describe the cloning of the eukaryotic recombinant PLDα of Vigna unguiculata (cowpea) into the yeast expression system Pichia pastoris and its two-step purification process. This allowed us to purify to homogeneity hundreds of micrograms of highly pure protein to conduct in fine structural studies.

Biochar and lignite affect H+-ATPase and H+-PPase activities in root tonoplast and nutrient contents of mung bean under salt stress.

This research was conducted to evaluate effects of biochar (50 and 100 g kg-1 soil) and lignite (50 and 100 g kg-1 soil) treatments on H+-ATPase and H+-PPase activity of root tonoplast, nutrient content, and performance of mung bean under salt stress. High saline conditions increased H+-ATPase and H+-PPase activities in root tonoplast, sodium (Na) content, reactive oxygen species (H2O2 and O2-) generation, relative electrolyte leakage (REL) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity in root and leaf, but decreased relative water content (RWC), chlorophyll content index, leaf area, potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn) and iron (Fe) content of plant tissues, root and shoot dry weight of mung bean. Lignite and biochar treatments decreased the H+-ATPase and H+-PPase activities of root tonoplast under salt stress. Moreover, these treatments increased the cation exchange capacity of soil and nutrient values in plant tissues. Biochar and lignite diminished the generation of reactive oxygen species and DPPH activity in root and leaf cells, and these superior effects improved chlorophyll content index, leaf area and growth of mung bean under both conditions. In general, the results of this study demonstrated that biochar and lignite decreased the entry of Na ion into the cells, enriched plant cells with nutrients, and consequently improved mung bean performance under salt toxicity.