Transcriptomic data reveals the dynamics of terpenoids biosynthetic pathway of fenugreek.

Medicinal plants are rich sources for treating various diseases due their bioactive secondary metabolites. Fenugreek (Trigonella foenum-graecum) is one of the medicinal plants traditionally used in human nutrition and medicine which contains an active substance, called diosgenin, with anticancer properties. Biosynthesis of this important anticancer compound in fenugreek can be enhanced using eliciting agents which involves in manipulation of metabolite and biochemical pathways stimulating defense responses. Methyl jasmonate elicitor was used to increase diosgenin biosynthesis in fenugreek plants. However, the molecular mechanism and gene expression profiles underlying diosgening accumulation remain unexplored. In the current study we performed an extensive analysis of publicly available RNA-sequencing datasets to elucidate the biosynthesis and expression profile of fenugreek plants treated with methyl jasmonate. For this purpose, seven read datasets of methyl jasmonate treated plants were obtained that were covering several post-treatment time points (6-120 h). Transcriptomics analysis revealed upregulation of several key genes involved in diosgenein biosynthetic pathway including Squalene synthase (SQS) as the first committed step in diosgenin biosynthesis as well as Squalene Epoxidase (SEP) and Cycloartenol Synthase (CAS) upon methyl jasmonate application. Bioinformatics analysis, including gene ontology enrichment and pathway analysis, further supported the involvement of these genes in diosgenin biosynthesis. The bioinformatics analysis led to a comprehensive validation, with expression profiling across three different fenugreek populations treated with the same methyl jasmonate application. Initially, key genes like SQS, SEP, and CAS showed upregulation, followed by later upregulation of Δ24, suggesting dynamic pathway regulation. Real-time PCR confirmed consistent upregulation of SQS and SEP, peaking at 72 h. Additionally, candidate genes Δ24 and SMT1 highlighted roles in directing metabolic flux towards diosgenin biosynthesis. This integrated approach validates the bioinformatics findings and elucidates fenugreek's molecular response to methyl jasmonate elicitation, offering insights for enhancing diosgenin yield. The assembled transcripts and gene expression profiles are deposited in the Zenodo open repository at https://doi.org/10.5281/zenodo.8155183 .

Differential responses of two fenugreek (Trigonella foenum-graecum L.) landraces pretreated with melatonin to prolonged drought stress and subsequent recovery.

BACKGROUND: Drought impairs growth, disturbs photosynthesis, and induces senescence in plants, which results in crop productivity reduction and ultimately jeopardizes human food security. The objective of this study was to determine major parameters associated with drought tolerance and recovery ability of fenugreek (Trigonella foenum-graecum L.), by examining differential biochemical and phenological responses and underlying enzyme activities as well as melatonin roles during drought stress and re-watering for two contrasting landraces. Moreover, the relative expression of three key genes involved in the biosynthesis pathway of diosgenin, including SQS, CAS, and BG, was investigated. RESULTS: Depending on the conditions, drought stress enhanced the activity of antioxidant enzymes and the osmoregulating compounds, non-enzymatic antioxidants, hydrogen peroxide content, and lipid peroxidation levels in most cases. Severe drought stress accelerated flowering time in Shushtar landrace (SHR) but had no significant effects on Varamin (VR). Pretreatment with melatonin delayed flowering time in SHR and caused high drought resistance in this landrace. Furthermore, melatonin significantly enhanced drought adaptability in VR by improving plant recovery ability. DISCUSSION: Based on our results plants' responses to drought stress and melatonin pretreatment were completely landrace-specific. Drought stress caused an increase in the relative expression of CAS gene and ultimately the accumulation of steroidal saponins in SHR. Melatonin compensated for the decrease in biomass production due to drought stress and finally increased steroidal saponins performance in SHR. Our study showed that melatonin can improve drought stress and recovery in fenugreek, but different factors such as genotype, melatonin concentration, and plant age should be considered.

Incorporation of compost and biochar enhances yield and medicinal compounds in seeds of water-stressed Trigonella foenum-graecum L. plants cultivated in saline calcareous soils.

BACKGROUND: The combination of compost and biochar (CB) plays an important role in soil restoration and mitigation strategies against drought stress in plants. In the current study, the impact of CB was determined on the characteristics of saline calcareous soil and the productivity of fenugreek (Trigonella foenum-graecum L.) plants. The field trials examined CB rates (CB0, CB10 and CB20 corresponding to 0, 10, and 20 t ha‒1, respectively) under deficit irrigation [DI0%, DI20%, and DI40% receiving 100, 80, and 60% crop evapotranspiration (ETc), respectively] conditions on growth, seed yield (SY), quality, and water productivity (WP) of fenugreek grown in saline calcareous soils. RESULTS: In general, DI negatively affected the morpho-physio-biochemical responses in plants cultivated in saline calcareous soils. However, amendments of CB10 or CB20 improved soil structure under DI conditions. This was evidenced by the decreased pH, electrical conductivity of soil extract (ECe), and bulk density but increased organic matter, macronutrient (N, P, and K) availability, water retention, and total porosity; thus, maintaining better water and nutritional status. These soil modifications improved chlorophyll, tissue water contents, cell membrane stability, photosystem II photochemical efficiency, photosynthetic performance, and nutritional homeostasis of drought-stressed plants. This was also supported by increased osmolytes, non-enzymatic, and enzymatic activities under DI conditions. Regardless of DI regimes, SY was significantly (P ≤ 0.05) improved by 40.0 and 102.5% when plants were treated with CB10 and CB20, respectively, as similarly observed for seed alkaloids (87.0, and 39.1%), trigonelline content (43.8, and 16.7%) and WP (40.9, and 104.5%) over unamended control plants. CONCLUSIONS: Overall, the application of organic amendments of CB can be a promising sustainable solution for improving saline calcareous soil properties, mitigating the negative effects of DI stress, and enhancing crop productivity in arid and semi-arid agro-climates.

Effect of salinity stress and surfactant treatment with zinc and boron on morpho-physiological and biochemical indices of fenugreek (Trigonella foenum-graecum).

Micronutrient application has a crucial role in mitigating salinity stress in crop plants. This study was carried out to investigate the effect of zinc (Zn) and boron (B) as foliar applications on fenugreek growth and physiology under salt stress (0 and 120 mM). After 35 days of salt treatments, three levels of zinc (0, 50, and 100 ppm) and two levels of boron (0 and 2 ppm) were applied as a foliar application. Salinity significantly reduced root length (72.7%) and shoot length (33.9%), plant height (36%), leaf area (37%), root fresh weight (48%) and shoot fresh weight (75%), root dry weight (80%) and shoot dry weight (67%), photosynthetic pigments (78%), number of branches (50%), and seeds per pod (56%). Fenugreek's growth and physiology were improved by foliar spray of zinc and boron, which increased the length of the shoot (6%) and root length (2%), fresh root weight (18%), and dry root weight (8%), and chlorophyll a (1%), chlorophyll b (25%), total soluble protein content (3%), shoot calcium (9%) and potassium (5%) contents by significantly decreasing sodium ion (11%) content. Moreover, 100 ppm of Zn and 2 ppm of B enhanced the growth and physiology of fenugreek by reducing the effect of salt stress. Overall, boron and zinc foliar spray is suggested for improvement in fenugreek growth under salinity stress.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Effect of hydroalcoholic extract of Trigonella foenum-graecum leaves on wound healing in type 1 diabetic rats.

Diabetes describes a group of metabolic disorders characterised by increased blood glucose concentration. People living with diabetes have a higher risk of morbidity and mortality than the general population. In 2015 it was estimated that there were 415 million (uncertainty interval: 340-536 million) people with diabetes aged 20-79 years, and 5.0 million deaths attributable to diabetes. When diabetic patients develop an ulcer, they become at high risk for major complications, including infection and amputation. The pathophysiologic relationship between diabetes and impaired healing is complex. Vascular, neuropathic, immune function, and biochemical abnormalities each contribute to the altered tissue repair. The use of herbal medicine has increased and attracted the attention of many researchers all over the world. In this study, we have evaluated the effect of 500mg/kg hydroalcoholic extract of Trigonella foenum-graecum leaves (TFG-E) on wound healing in diabetic rats using a full-thickness cutaneous incisional wound model. Wounds of treated animals showed better tensiometric indices, accelerated wound contraction, faster re-epithelialisation, improved neovascularisation, better modulation of fibroblasts and macrophage presence in the wound bed and moderate collagen formation.

Soil-applied Nickel Generates Differential Responses in Growth, Physiology and Oxidative Metabolism of Fenugreek (Trigonella foenum-graecum L.) Varieties.

This work aims to evaluate the potential of nickel (Ni), an essential micronutrient, as an oxidative stress inducer along with associated morphological and biochemical responses in different varieties of fenugreek (Trigonella foenum-graecum L.), a chief economically cultivated crop of India. Varietal differences in crop performance upon exposure to 0, 20, 40, 60 and 80 mg Ni kg- 1 soil reflects that Ni applied at 20 mg Ni kg- 1 soil offers growth-promoting effects, improved photosynthesis attributes, carbonic and nitrate reductase activities more profound in PEB followed by AFg2, AFg1 and UM185 variety. This study observed a dose-dependent reduction in all the above parameters. Maximum toxic effects were noticed at 80 mg kg- 1 Ni, manifested in the form of enhanced H2O2 and MDA contents, which were efficiently counteracted by augmentation in proline content, SOD, POX, CAT and APX activities in PEB over other varieties, suggesting that the Ni tolerance in fenugreek varieties can be organized as PEB > AFg2 > AFg1 > UM185.

Hepatorenal Protective Effect of Fenugreek Aqueous Extract against Lead Toxicity in Experimental Rats.

In this study, aqueous extract of germinated fenugreek seeds was investigated to assess its therapeutic effect on hepatorenal lead toxicity in experimental rats. After overnight fasting, rats were injected intraperitoneally with 0.5 mL of lead acetate at a dose of 35 mg/kg body weight for five consecutive days. Animals were divided into four groups of ten rats each: normal control; untreated negative control and rats treated with 200 or 400 mg/kg body weight of the aqueous extract. Treatments were performed by intraperitoneal injection of 1mL of the extract once a day for 28 consecutive days. Results showed significant differences between treated and control groups during the whole period of the experiment. This was demonstrated by improving body weight and level of serum total protein, decreasing levels of serum ALT, AST, total bilirubin, blood urea nitrogen, and creatinine. As well, histological analysis revealed a marked reduction in inflammation and structural alterations of liver and kidney organs of fenugreek-treated rats. This hepatoprotective effect can be attributed to the anti-inflammatory, anti-oxidant and regenerative capacity of the high content of the phytochemical constituents in the extract.

[Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes].

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 µg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Effect of Elicitation with Iron Chelate and Sodium Metasilicate on Phenolic Compounds in Legume Sprouts.

Seven-day-old sprouts of fenugreek (Trigonella foenum-graecum L.), lentil (Lens culinaris L.), and alfalfa (Medicagosativa L.) were studied. The legume seeds and then sprouts were soaked each day for 30 min during 6 days with water (control) or mixture of Fe-EDTA and sodium silicate (Optysil), or sodium silicate (Na-Sil) alone. Germination and sprout growing was carried out at temperature 20 ± 2 °C in 16/8 h (day/night) conditions. Phenolic compounds (free, ester, and glycosides) content were determined by HPLC-ESI-MS/MS using a multiple reaction monitoring of selected ions. Flavonoids and phenolic acids were released from their esters after acid hydrolysis and from glycosides by alkaline hydrolysis. The presence and high content of (-)-epicatechin (EC) in fenugreek sprouts was demonstrated for the first time. Applied elicitors decreased the level of free EC in fenugreek and alfalfa sprouts but enhanced the content of its esters. Besides, elicitors decreased the content of quercetin glycosides in lentil and fenugreek sprouts but increased the content of quercetin and apigenin glycosides in alfalfa sprouts. The applied elicitors decreased the glycoside levels of most phenolic acids in lentil and p-hydroxybenzoic acid in fenugreek, while they increased the content of this acid in alfalfa. The mixture of iron chelate and sodium silicate had less effect on changes in flavonoid and phenolic acid content in legume sprouts than silicate alone. In general, the used elicitors increased the content of total phenolic compounds in fenugreek and alfalfa sprouts and decreased the content in lentil sprouts. Among the evaluated elicitors, Optysil seems to be worth recommending due to the presence of iron chelate, which can be used to enrich sprouts with this element.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

Comparative Transcriptome Analysis Identifies Genes Involved in Diosgenin Biosynthesis in Trigonella foenum-graecum L.

Trigonella foenum-graecum L. (fenugreek) is a valuable resource of producing diosgenin which serves as a substrate for synthesizing more than two hundred kinds of steroidal drugs. Phytochemical analysis indicated that methyl jasmonate (MeJA) efficiently induced diosgenin biosynthesis in fenugreek seedlings. Though early steps up to cholesterol have recently been elucidated in plants, cytochrome P450 (CYP)- and glycosyltransferase (GT)-encoding genes involved in the late steps from cholesterol to diosgenin remain unknown. This study established comparative fenugreek transcriptome datasets from the MeJA-treated seedlings and the corresponding control lines. Differential gene expression analysis identified a number of MeJA-induced CYP and GT candidate genes. Further gene expression pattern analysis across a different MeJA-treating time points, together with a phylogenetic analysis, suggested specific family members of CYPs and GTs that may participate in the late steps during diosgenin biosynthesis. MeJA-induced transcription factors (TFs) that may play regulatory roles in diosgenin biosynthesis were also discussed. This study provided a valuable genetic resource to functionally characterize the genes involved in diosgenin biosynthesis, which will push forward the production of diosgenin in microbial organisms using a promising synthetic biology strategy.

Trace Metal Accumulation in Trigonella foenum-graecum Irrigated with Wastewater and Human Health Risk of Metal Access Through the Consumption.

The aim of the present research was to determine the trace metal accumulations in Trigonella foenum-graecum irrigated with three different water regimes (ground water, canal water and sugar mill water). Also, transfer factors, pollution load indices, and health risk indices were assessed to evaluate metal transport and accumulation through the food chain. The analysis was conducted by Atomic Absorption Spectrophotometer (Shimadzu model AA-6300) to evaluate the concentration of metals in water, soil and vegetables. Trace metal concentrations in water samples ranged from 0.84 to 1.67, 0.42 to 0.72, 0.45 to 0.85, 2.51 to 9.99, 1.21 to 1.92, 1.82 to 9.98 and 0.64 to 0.91 mg/kg for Cd, Cr, Cu, Fe, Ni, Zn and Mn, respectively. The mean metal concentrations in soil samples were determined as 0.25, 0.86, 0.96, 3.37, 0.4, 0.44 and 2.31 mg/kg for Cd, Cr, Cu, Fe, Ni, Zn and Mn, respectively. Trace metal accumulations in T. foenum-graecum samples gathered from where soil samples were taken are as follows: The contents of Cd, Cr, Cu, Fe, Ni, Zn and Mn ranged from 0.48 to 1.06, 0.11 to 0.35, 0.15 to 0.29, 1.43 to 8.39, 0.39 to 0.54, 2.1 to 3.05 and 0.42 to 0.47 mg/kg, respectively. Statistical analyses showed that the treatments have non-significant effect (p > 0.05) on concentrations of metals in T. foenum-graecum samples collected from three sites for Ni, Cr, Cu, Zn and Mn and significant effect on Fe and Cd.

Zinc Oxide Nanoparticles Induced Oxidative Stress and Changes in the Photosynthetic Apparatus in Fenugreek (Trigonella foenum graecum L.).

The aim of this work was to study the toxicity of nanosheet zinc oxide nanoparticle with the size of 45 nm. The penetration of nanoparticles at an exposure by a localized spray does not make it possible to understand the mechanism of transport and bioavailability of the nanoparticles. In contrast, nanoparticles penetrated, via the roots, as a function of their diameter, the smaller ones having caused leaf stress (by translocation) at low concentrations. So that the choice of method of root application. Plants treated with 50 mg L-1 of ZnO-NPs presented disturbance in leaf due to changes in chlorophyll's biosynthesis. The highest value of the photosynthetic pigments was recorded at 5 mg L-1 of ZnO-NPs. However, the treatment with 50 mg L-1 of ZnO-NPs caused a decrease in the levels of chlorophyll a and b. Moreover, ZnO-NPs leaves significantly enhanced antioxidant enzymes activities.

Reserve Mobilization, Membrane Damage and Solutes Leakage in Fenugreek Imbibed with Urban Treated Wastewater.

The study aimed to determine the impact of treated domestic wastewater on seed germination, seedling growth and reserve mobilization from Fenugreek (Trigonellafoenum graecum L.). Seeds were germinated by soaking in distilled water (H2O) or wastewater treated with various methods: activated sludge processes (T1), facultative lagoons treatment (T2) and dilution. Results show high levels of organic matter (OM), suspended solids (TSS) and nutrients in TWW (T2) than that of TWW (T1). The embryo length and biomass of fenugreek imbibed by TWW were higher in TWW (T2) compared with TWW (T1). There was more reduction in free amino acids and soluble sugar contents in fenugreek treated with TWW (T1) than treated by TWW (T2). Important solutes leakage is recorded by measuring electric conductivity during seed imbibition with TWW. Improving the quality of wastewater by dilution (50%) stimulated germination of seeds and the growth of the tested plant. Moreover, it significantly reduces the solutes leakage and enhanced seed metabolites accumulation.

Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata L.).

Improving fish defense through the preventive administration of immunostimulants has an important role in controlling the outbreak of the disease in aquaculture. As a continuity of our previous studies, this paper describes the effects of dietary fenugreek (Trigonella foenum graecum) seeds on the skin mucosal antioxidant status and immune response of gilthead seabream (Sparus aurata L.). Fish were fed with four experimental diets: one a basal diet (control) and three diets with powdered fenugreek seeds incorporated in the fish feed at 1%, 5%, and 10%. After eight weeks of feeding, free radicals scavenging and antioxidant assays were assessed in skin mucus by measuring the peroxidation of phospholipid liposomes, hydroxyl radical and hydrogen peroxide scavenging, measurement of total antioxidant activity and the determination of antioxidant activity in a linoleic acid system. The skin mucosal immune response was evaluated by measuring the IgM levels and some enzymatic activities (peroxidase, antiprotease, protease, esterase, and ceruloplasmin). Our results demonstrated that fenugreek inclusion improved the hydroxyl radical scavenging capacity and conferred very high antioxidant activity. Besides, only the highest supplementation level (10%) was able to augment the peroxidase and protease activities confronted by a general decrement in the antiprotease activity in the experimental fed groups with 1% and 10%. These results suggest that the dietary administration of fenugreek at the higher inclusion dose enhances the skin mucosal immunity response and the antioxidant status of gilthead seabream a species with one of the highest rates of production in marine aquaculture.

Effect of planting time and vermicompost on the proteomic pattern of fenugreek (Trigonella foenum-graecum)

The fenugreek is one of the most important medicinal plants belongs to Fabaceae, originated in West Asia, Iran and Mediterranean regions. This research included a qualitative study of fenugreek proteins using SDS-PAGE electrophoresis on polyacrylamide gel and the separation of protein bands of fenugreek leaves in different treatments of vermicompost fertilizer and cultivating dates. Results showed that a band (about 80 kDa) on the first planting date (May 31) is observed in all samples except for sample a1 (10 t/ha vermicompost on May 31). Another significant difference was the band contained in the third planting date (31 September) and in the molecular weight of about 15 kDa, which was not seen in other dates. This difference can be due to the synthesis of this protein with the mentioned weight under the conditions of reducing the temperature in the early fall. It also showed more differences in two-dimensional electrophoresis, for example, in 14 kDa and PI in the range of 4.5-4.7 in treatment without fertilizer, no protein expression was observed, which was consistent with the results of the SDS-PAGE test.

Regulation of Mitochondrial and Cytosol Antioxidant Systems of Fenugreek (Trigonella foenum graecum L.) Exposed to Nanosized Titanium Dioxide.

In the present study, the interactions between nanoparticle (NP) exposure, root application and plants were examined. NPs are potentially responsible for conformational changes in polysaccharides, lipids, proteins, pectin, suberin and lignin molecules. 4 days of treatment with metal oxide caused a statistically significant increase in nicotinamide adénine dinucléotide oxidase activity in mitochondria and cytosol. Following exposure to TiO2NP, even lipid peroxidation levels decreased in the mitochondria (leaves, stem and root) and in the cytosol (leaves and root), although it increased in the cytosol of the stem. Malondialdehyde accumulation was found to be higher in the cytosol compared to the mitochondria of stems, and in the cytosol of leaves and roots. NPs caused alterations in metabolism, antioxidant enzyme activities (guaiacol peroxidase, catalase and ascorbate peroxidase) and the generation of oxidative stress. Effects caused by exposures to NPs were influenced by differences in metabolic responses in plant parts, plant compartments, the period of exposure and the NP doses.

Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum).

MAIN CONCLUSION: Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed. Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the next-generation sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases. Within the transcripts related to sterol and steroidal saponin biosynthesis, we discovered novel candidate genes of diosgenin biosynthesis and validated their expression using quantitative RT-PCR analysis. Based on these findings, we supported the idea that diosgenin is biosynthesized from cycloartenol via cholesterol. This is the first report on the next-generation sequencing of cDNA-RDA products. Analysis of the transcriptomes enriched in low copy sequences contributed substantially to our understanding of the biochemical pathways of steroid synthesis in fenugreek.

Quality and antioxidant response of gilthead seabream (Sparus aurata L.) to dietary supplements of fenugreek (Trigonella foenum graecum) alone or combined with probiotic strains.

The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream.