Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways.

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with ß-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans.

Biochemical and Anatomical Investigation of Sesbania herbacea (Mill.) McVaugh Nodules Grown under Flooded and Non-Flooded Conditions.

Sesbania herbacea, a native North American fast-growing legume, thrives in wet and waterlogged conditions. This legume enters into symbiotic association with rhizobia, resulting in the formation of nitrogen-fixing nodules on the roots. A flooding-induced anaerobic environment imposes a challenge for the survival of rhizobia and negatively impacts nodulation. Very little information is available on how S. herbacea is able to thrive and efficiently fix N2 in flooded conditions. In this study, we found that Sesbania plants grown under flooded conditions were significantly taller, produced more biomass, and formed more nodules when compared to plants grown on dry land. Transmission electron microscopy of Sesbania nodules revealed bacteroids from flooded nodules contained prominent polyhydroxybutyrate crystals, which were absent in non-flooded nodules. Gas and ion chromatography mass spectrometry analysis of nodule metabolites revealed a marked decrease in asparagine and an increase in the levels of gamma aminobutyric acid in flooded nodules. 2-D gel electrophoresis of nodule bacteroid proteins revealed flooding-induced changes in their protein profiles. Several of the bacteroid proteins that were prominent in flooded nodules were identified by mass spectrometry to be members of the ABC transporter family. The activities of several key enzymes involved in nitrogen metabolism was altered in Sesbania flooded nodules. Aspartate aminotransferase (AspAT), an enzyme with a vital role in the assimilation of reduced nitrogen, was dramatically elevated in flooded nodules. The results of our study highlight the potential of S. herbacea as a green manure and sheds light on the morphological, structural, and biochemical adaptations that enable S. herbacea to thrive and efficiently fix N2 in flooded conditions.

Influence of direct and sequential extraction methodology on metabolic profiling.

A systematic comparison was made of the detected metabolite profiles for two plant materials (black beans and soybeans) and a dietary supplement (black cohosh) extracted using sequential (hexane, ethyl acetate, and 50% aqueous methanol) and direct extraction with three solvent systems (80% aqueous methanol, methanol/chloroform/water (2.5:1:1, v/v/v) and water). Extracts were analyzed by LC-MS (without derivatization) and GC-FID (with BSTFA/TMCS derivatizations). For sequential extraction, HPLC-UV and BSTFA/TMCS-derivatized GC-FID detection were more responsive to the polar molecules with a rough distribution of 10%, 10%, and 80% of the total signals in hexane, ethyl acetate, and 50% aqueous methanol, respectively. With HPLC-MS detection, the distribution of signals was more balanced, roughly 40%, 30%, and 30% for the same extracts (hexane, ethyl acetate, and 50% aqueous methanol). For direct extraction, HPLC-UV and BSTFA/TMCS-derivatized 4GC-FID provided signals between 60% and 150% of the total sequential extracted signals. The overlap of signals for the 3 sequential extracts ranged from 1% to 3%. The overlap of the signals for direct extraction with the total for sequential extraction ranged from 15% to 98%. With HPLC-MS detection, signals varied from 30% to 40% of the total signals for sequential extraction. Multivariate analysis showed that the components for the sequential and direct extracts were statistically different. However, each extract, sequential or direct, allowed discrimination between the 3 plant materials.

Extreme effects of Seabuckthorn extracts on influenza viruses and human cancer cells and correlation between flavonol glycosides and biological activities of extracts.

Seabuckthorn is a medicinal plant that is used to prevent cold. It was tested for its metabolic content followed by activity against cancer and virus. The metabolic distribution of different polarity solvent extractions from the leaves was analyzed by LC-MS/MS. Flavonol glycoside contents in EA and Bu extracts were higher than MeOH and DW was observed. MeOH and EA extracts recorded high activity against influenza A/PR virus with IC50 of 7.2 µg/mL and 10.3 µg/mL compared with known drug Oseltamivir of 60.3 µg/mL. A similar trend showed in influenza A/Victoria virus. In case of influenza B viruses such as B/Lee and B/Maryland, EA extract (2.87 µg/mL and 4.5 µg/mL of IC50) emerged strongest among other extracts and Oseltamivir (103.73 µg/mL and 71.6 µg/mL). Each extract showed potent anticancer activities. Interestingly, Bu extract showed stronger anticancer activity against human cancer cells such as NCL-H1299, HeLa, SKOV and Caski (8.2 µg/mL, 8.6 µg/mL, 18.2 µg/mL and 9.2 µg/mL of IC50) respectively. Correlation study reveals that aglycones and flavonol mono-glycosides highly correlated with anti-influenza activities but not correlated with anticancer activities. Reversely, di-glycosides and tri-glycosides have a high correlation with cytotoxic effect with both normal and cancer cells. Therefore, this study provides significant information concerning Seabuckthorn for further medicinal drug development.

Proteomic analysis of anti-nutritional factors (ANF's) in soybean seeds as affected by environmental and genetic factors.

The genotype (G), environment (E), and the relationship between G and E on soybean seed anti-nutritional factors (ANF's) were examined under three different agro-climatic conditions. The field trials were conducted at Maryland, South Carolina and South Dakota using nine region specific genotypes. At each location, the nine genotypes were grown with two planting/sowing dates. Differentially expressed protein spots from the two-dimensional gel electrophoresis were analyzed using mass spectrometry. Seven ANF's corresponding to soybean agglutinin and Kunitz trypsin inhibitor were identified based on the statistical significance levels at p<0.005. The G and E conditions (planting/sowing season) influences the ANF's content. This initial study suggests that early sowing reduces the total ANF's content irrespective of genotypes and their growing locations.

Metabolite changes in nine different soybean varieties grown under field and greenhouse conditions.

Global food security remains a worldwide concern due to changing climate, increasing population, and reduced agriculture acreages. Greenhouse cultivation increases productivity by extending growing seasons, reducing pest infestations and providing protection against short term drastic weather fluctuations like frost, heat, rain, and wind. In the present study, we examined and compared the metabolic responses of nine soybean varieties grown under field and greenhouse conditions. Extracts were assayed by GC-FID, GC-MS, and LC-MS for the identification of 10 primary (amino acids, organic acids, and sugars) and 10 secondary (isoflavones, fatty acid methyl esters) metabolites. Sugar molecules (glucose, sucrose, and pinitol) and isoflavone aglycons were increased but the isoflavones glucoside content decreased in the greenhouse cultivated soybeans. The amino acids and organic acids varied between the varieties. The results show that clustering (PCA and PLS-DA) patterns of soybean metabolites were significantly influenced by the genetic variation and growing conditions.

Genetic diversity of UPASI tea clones (Camellia sinensis (L.) O. Kuntze) on the basis of total catechins and their fractions.

Tea leaf catechins and the ratio of dihydroxylated to trihydroxylated catechin fractions were analysed to identify the genetic diversity of 26 UPASI released tea clones. Principal component analysis (PCA) based on regression factor separated tea clones into five groups according to their jats (Jats are region based rays for e.g., Assam, China and Cambod origin) as well as their quality constituents (such as total polyphenols, total catechins, amino acids in the green leaves and liquor characteristics of black tea), particularly the catechins. Group 1 represented medium quality (quality of the final produce) clones, such as UPASI-10, UPASI-12 and UPASI-15 and drought tolerant clones like UPASI-1, UPASI-2, UPASI-9 and UPASI-10. Group 2 contained purely "China" cultivars while group 3 possessed high quality tea cultivars. "Assam" (group 5) teas had the lowest ratio of dihydroxylated to trihydroxylated catechin fractions (1:4) than the "Chinery" (group 2) teas (1:5). This biochemical differentiation indicated that there is a vast genetic diversity in UPASI released tea clones in terms of catechin fractions, even though the majority of them were selected from one tea estate located in the Nilgiris.

Spectroscopic determination of metabolic and mineral changes of soya-chunk mediated by Aspergillus sojae.

Time dependent changes of primary (GC-MS), isoflavones (LC-MS) and minerals (ICP-OES) content of fermented soya-chunk were compared with un-fermented (0H) soya-chunk and presented. Results revealed that the amino acid content increased gradually based on the fermentation time; whereas the maltose, sucrose and fructose contents were reduced due to the fungal growth. The glucosides changed extensively during the initial fermentation time resulting in augmentation of aglycones and phytoalexins. This affects the antioxidant potential whereas the DPPH and ABTS of 0H showed lowest activity (18.15% and 54.92%) and increased quite high with fungal fermentation (45.81% and 93.47%). The calcium (0.55%), magnesium (0.47 mg/kg), nickel (5.17 mg/kg l(-1)), and copper (8.33 mg/kg l(-1)) content were increased during the fermentation and in a decrease of iron and aluminium contents. Findings suggest that the soya-chunk prepared by fungal fermentation will improve the antioxidant and mineral content and hence their nutritional property will be enhanced for humans.

Anti-influenza (H1N1) potential of leaf and stem bark extracts of selected medicinal plants of South India.

Variations in antioxidant and anti-viral activities (against Influenza AP/R/8 (H1N1) virus) between the leaves and stem bark of selected medicinal plants were studied. Malin Darby canine kidney (MDCK) cells were used for the viral infection and the antiviral activity of the extracts was studied using sulphorhodamine B (SRB) assay. The stem bark of the plants including Strychnos minor, Diotacanthus albiflorus, Strychnos nux-vomica and Chloroxylon swietenia showed higher flavonoid contents as well as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging activity when compared with their leaves. In case of 1,1-diphenyl-2-picrylhydrazyl (DPPH) activity, the stem bark of S. nux-vomica and leaf extract of C. swietenia showed the highest activity. Based on the IC50 values, the stem bark extracts of Cayratia pedata (20.5 µg/mL) and S. minor (22.4 µg/mL) showed high antiviral activity. In the mean-time S. nux-vomica, C. swietenia and C. swietenia bark extracts showed cytotoxicity to the MDCK cells. When comparing the stem bark and leaves the content of gallic acid, ferulic acid, o-coumaric acid, total flavonoids (TFC) and total phenols (TPC) was higher in stem bark and hence their anti-viral activity was high. Further study based on the metabolites against H1N1 can reveal the potential of therapeutic compounds against the viral disease.

Screening of ethnic medicinal plants of South India against influenza (H1N1) and their antioxidant activity.

Antiviral activity against H1N1 influenza was studied using ethnic medicinal plants of South India. Results revealed that Wrightia tinctoria (2.25 µg/ml) was one of the best antidotes against H1N1 virus in terms of inhibitory concentration of 50% (IC50) whereas the control drug Oseltamivir showed 6.44 µg/ml. Strychnos minor, Diotacanthus albiflorus and Cayratia pedata showed low cytotoxicity (>100) to the MDCK (Malin darby canine kidney) cells by cytotoxicity concentration of 50% (CC50) and possessed antiviral activity suggesting that these plants can be used as herbal capsules for H1N1 virus. W. tinctoria and S. minor showed high therapeutic indexes (TI) such as 12.67 and 21.97 suggesting that those plants can be used for anti-viral drug development. The CC50 values of Eugenia singampattiana (0.3 µg/ml), Vitex altissima (42 µg/ml), Salacia oblonga (7.32 µg/ml) and Salacia reticulata (7.36 µg/ml) resulted in cytotoxicity of the MDCK cells, due to their high phenolic content. Findings from this study state that the plant W. tinctoria can be a potent source for third generation anti-viral drug development against H1N1.

Metabolic variation and antioxidant potential of Malus prunifolia (wild apple) compared with high flavon-3-ol containing fruits (apple, grapes) and beverage (black tea).

Secondary metabolic variation of wild apple (Malus prunifolia) was compared with fruits that contained high flavan-3-ol like grapes (GR), apple (App) and the beverage, black tea (BT). The polyphenol contents in wild apple was higher than in GR and App but less than BT. The identified phenolic acids (gallic, protocatechuic, chlorogenic, p-coumaric and ferulic acids) and flavonoids (quercetin and myricetin) indicate that wild apple was higher than that of App. Among all the samples, BT had highest antioxidant potential in terms of 2,2'-Azinobis (3-thylbenzothiazoline-6-sulfonic acid) diammonium salt (95.36%), metal chelating (45.36%) and phosphomolybdenum activity (95.8 mg/g) because of the high flavan-3-ol content. The gallic acid and epigallocatechin gallate were highly correlated with antioxidant potential and these metabolites levels are higher in wild apple than that of App. Wild apples being a non-commercial natural source, a detailed study of this plant will be helpful for the food additive and preservative industry.