Characterization, inhibitory activity and mechanism of polyphenols from faba bean (gallic-acid and catechin) on α-glucosidase: insights from molecular docking and simulation study.

The chemo-profiling of ethanolic extract of faba beans seeds was performed and explored as an α-glucosidase inhibitor. The inhibition of α-glucosidase is one of the alternatives approach to control postprandial hyperglycemia by, resulting in the delay of the carbohydrate digestion of absorbable monosaccharides. Ethanolic seed extract showed phenolic compounds, flavonoid such as gallic acid (m/z [M- H] = 169.0124,C7H6O5) ellagic acid derivatives epigallocatechin (m/z [M- H = 305.0644,C15H14O7),catechin (m/z [M- H] = 289.0656,C15H14O6), epigallocatechin gallate (m/z [M- H] = 457.0578,C22H18O11) and epicatechin monogallate (m/z [M- H] = 441.081, C22H18O10). The extract was found to exert inhibitory activity (88.28 ± 2.67%) (IC50 value of 2.30 ± 0.032 mg/mL) with a mixed mode of inhibition (Km, apparent = 0.54 ± 0.020 mM and Vmax, apparent 0.136 ± 0.04 mM/min). Molecular docking studies of gallic acid and catechin on α-glucosidase proposed productive binding modes having binding energy (-6.58 kcal/mol and -7.25 kcal/mol) with an effective number of hydrogen bonds and binding energy. Tyr63, Arg197, Asp198, Glu 233, Asn324, Asp 326 of α-glucosidase participated in binding events with gallic acid and catechin. Molecular dynamics simulation studies were performed for both complexes i.e. gal:α-glucosidase and cat:α-glucosidase along with apo state of α-glucosidase, which revealed stable systems during the simulation. These findings of the present study may give an insight into the further development of the novel antidiabetic drug from the seeds of faba beans.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

Hybrid embryos of Vicia faba develop enhanced sink strength, which is established during early development.

Selfed and crossed seeds of two homozygous Vicia faba lines served as models for the analysis of the physiological and molecular mechanisms underlying embryo heterosis. Profiles of transcripts, metabolites and seed contents of developing embryos were analysed to compare the means of reciprocally crossed and selfed seeds growing on the same mother plants. The mean weight of mature hybrid seeds was demonstrably higher, revealing mid-parent heterosis. Hybrid embryos exhibited a prolonged early phase of development and delayed onset of storage activity. Accordingly, transcript profiling indicates stimulation of cell proliferation, an effect, which is potentially mediated by activation of auxin functions within a framework of growth-related transcription factors. At the transcript level, activated cell proliferation increased assimilate uptake activity and thereby seed sink strength. This situation might finally lead to the increased size of the hybrid seeds. We conclude that hybrid seeds are characterised by accelerated growth during early development, which increases storage capacity and leads to higher metabolic fluxes. These needs are, at least partially, met by increased assimilate uptake capacity. The stimulated growth of hybrid seeds shifted metabolite profiles and potentially depleted available pools. Such metabolic shifts are most likely secondary effects resulting from the higher storage capacity of hybrid seeds, a heterotic feature, which is itself established very early in seed development.

Quantitative genetic analysis of embryo heterosis in faba bean (Vicia faba L.).

Seeds, i.e. embryos, may be genetically different from either of their parents and moreover may express their own heterosis. The objective was to genetically analyse embryo heterosis for their own weight (i.e. seed weight) in comparison with their seedlings' heterosis, taking the large-seeded crop (Vicia faba L.) as model. A specific diallel mating scheme was used, based on four parental lines, creating 76 seed genotypes in generations P, F(1), F(2) and BC. Mature seed weight was assessed for these embryo genotypes in 3 years at one German location, and young plant biomass yield of seedlings emerging from these seeds in two greenhouse experiments. The quantitative genetic analysis showed an average of 10.6% mid-parent heterosis for mature seed weight and 14.5% mid-parent heterosis for juvenile biomass. In both traits, the embryos contributed markedly and significantly via their own genes to the genetic variation. For mature embryo weight heterosis, apparently the parental difference in seed weight was decisive, whereas for juvenile biomass heterosis, genetic unrelatedness of parents had priority.

Lead-contaminated soil induced oxidative stress, defense response and its indicative biomarkers in roots of Vicia faba seedlings.

Seeds of Vicia faba. L were grown in increasing concentrations of lead (Pb)-added soils (0-2,000 mg/kg). After germination of 25 days, roots were harvested to investigate oxidative stress, defense response and indicative biomarkers based upon chemical analyses and biological measurements. The results showed that higher concentrations of Pb-polluted soils led to seedling growth inhibition, indicative of phytotoxicity. O (2) (*-) and lipid peroxidation were increased with the increase of available Pb in soils and Pb contents in roots, displaying a "J"-shaped dose response curve, whereas H(2)O(2) showed a biphasic dose response curve (a consecutive "J"-shaped and inverted "U"-shaped curve). Superoxide dismutase (SOD), guaiacol peroxidase (POD) and ascorbate peroxidase (APX) enzymes were activated by soil Pb, displaying biphasic curves. The upregulated POD and APX enzymes might be major scavengers of excessive H(2)O(2) when CAT activities were drastically reduced with the increasing soil Pb. The enhanced glutathione (GSH) and APX activities suggested that GSH-ascorbate cycle also participated in eliminating H(2)O(2). Moreover, obvious changes were observed in SOD, CAT and POD isoenzyme patterns, but not in APX except increasing intensities of bands. HSP70 synthesis was significantly induced by extraneous Pb from 125 to 1,000 mg/kg and showed a biphasic curve in this experiment. Comparatively, HSP70 and lipid peroxidation might be more sensitive than other parameters in response to Pb stress, suggesting that these two parameters in the roots might be potential biomarkers for early bioassay of Pb-contaminated soils.

Purification of alpha-galactosidase by affinity precipitation with alginate.

Affinity precipitation is a technique which is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation. It is a relatively simple, convenient, and reproducible technique that results in high target molecule recovery at high specificity. We describe, here, an efficient and rapid purification procedure for Vicia faba alpha-galactosidase (EC 3.2.1.22) by using affinity precipitation with alginate. The enzyme was purified with 43% activity yield and 40-fold purification. SDS-PAGE of the purified enzyme showed a single band and a subunit weight of 44 kDa. The properties of the enzyme were also searched. The results showed that the general properties of the enzyme offer potential for use of this alpha-galactosidase in several production processes.

Establishment of the regeneration system for Vicia faba L.

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba. Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

Genetic variation among Egyptian cultivars of Vicia faba L.

Nine Egyptian cultivars of Vicia faba were analyzed using electrophoretic and quantitative data from seed albumins and globulins and 100 seeds weight to measure genetic variation among faba bean cultivars in Egypt. Wide genetic variation was indicated for all the traits studied. The elctrophoregrams showed identity profile for each cultivar supporting the validity of electrophoresis of seed protein components in cultivar identification and assessing genetic variation in Vicia faba and other out-breeding plants at the infra-specific level. Negative correlation was indicated between seed globulins and both seed albumins and seed weight. Principal component analysis and cluster analyses indicated higher role of seed albumins over seed globulins in genetic variation within Vicia faba in Egypt.

Induction of wall ingrowths of transfer cells occurs rapidly and depends upon gene expression in cotyledons of developing Vicia faba seeds.

Abaxial epidermal cells of developing faba bean (Vicia faba) cotyledons are modified to a transfer cell morphology and function. In contrast, the adaxial epidermal cells do not form transfer cells but can be induced to do so when excised cotyledons are cultured on an agar medium. The first fenestrated layer of wall ingrowths is apparent within 24 h of cotyledon exposure to culture medium. The time course of wall ingrowth formation was examined further. By 2 h following cotyledon excision, a 350 nm thick wall was deposited evenly over the outer periclinal walls of adaxial epidermal cells and densities of cytoplasmic vesicles increased. After 3 h in culture, 10% of epidermal cells contained small projections of wall material on their outer periclinal walls. Thereafter, this percentage rose sharply and reached a maximum of 90% by 15 h. Continuous culture of cotyledons on a medium containing 6-methyl purine (an inhibitor of RNA synthesis) completely blocked wall ingrowth formation. In contrast, if exposure to 6-methyl purine was delayed for 1 h at the start of the culture period, the adaxial epidermal cells were found to contain small wall ingrowths. Treating cotyledons for 1 h with 6-methyl purine at 15 h following cotyledon excision halted further wall ingrowth development. We conclude that transfer cell induction is rapid and that signalling and early events leading to wall ingrowth formation depend upon gene expression. In addition, these gene products have a high turnover rate.

Comparative analysis of mitotic aberrations induced by diethyl sulphate (DES) and sodium azide (SA) in Vicia faba L. (Fabaceae).

The present investigation provides a comparative account of different concentrations (0.01, 0.02, 0.03, 0.04, 0.05 and 0.06%) of diethylsulphate (DES) and Sodium Azide (SA) on mitotic aberrations, seed germination, seedling survival, plant height and mitotic index in Vicia faba L. variety major. The control plants were normal while as treated ones showed significant alterations. The mutagens caused dose dependent decrease in seed germination, seedling survival, plant height and mitotic index. All the parameters were found negatively affected and were positively correlated with mutagenic concentrations. The cytological study revealed various types of mitotic aberrations, among them the dominant were fragments, stickiness, precocious separation, c-metaphase, ring chromosomes, unequal separation, laggards, bridges, micronuclei, disturbed anaphase etc. Stickiness and fragments were more frequent as compared to other types.

Energy status and its control on embryogenesis of legumes: ATP distribution within Vicia faba embryos is developmentally regulated and correlated with photosynthetic capacity.

To analyse the energy status of Vicia faba embryos in relation to differentiation processes, we measured ATP concentrations directly in cryosections using a quantitative bioluminescence-based imaging technique. This method provides a quantitative picture of the ATP distribution close to the in vivo situation. ATP concentrations were always highest within the axis. In pre-storage cotyledons, the level was low, but it increased strongly in the course of further development, starting from the abaxial region of cotyledons and moving towards the interior. Greening pattern, chlorophyll distribution and photosynthetic O2 production within embryos temporally and spatially corresponded to the ATP distribution, implicating that the overall increase of the energy state is associated to the greening process. ATP patterns were associated to the photosynthetic capacity of the embryo. The general distribution pattern as well as the steady state levels of ATP were developmentally regulated and did not change upon dark/light conditions. The major storage protein legumin started to accumulate in abaxial regions with high ATP, whereas starch localized in regions with relatively lower ATP levels. This suggests a role of the energy state in the partitioning of assimilates into the different storage-product classes. Highest biosynthetic rates occurred when cotyledons became fully green and contained high ATP levels, implicating that a photoheterotrophic state was required to ensure high fluxes. Based on these data, we propose a model for the role of embryonic photosynthesis to improve the energy status of the embryo.

Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes.

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O(2) levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O(2) concentration (1.1 micro M). Embryo respiration was more strongly inhibited by low O(2) during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O(2) limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O(2) and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O(2) production elevated the internal level up to approximately 50% of atmospheric O(2) concentration (135 micro M). Upon light conditions, embryos partitioned approximately 3-fold more [(14)C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O(2) supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O(2) and energy that is readily used for biosynthesis and respiration.