Simultaneously Determined Antioxidant and Pro-Oxidant Activity of Randomly Selected Plant Secondary Metabolites and Plant Extracts.

Oxidative stress is a well-known phenomenon arising from physiological and nonphysiological factors, defined by the balance between antioxidants and pro-oxidants. While the presence and uptake of antioxidants are crucial, the pro-oxidant effects have not received sufficient research attention. Several methods for assessing pro-oxidant activity, utilizing various mechanisms, have been published. In this paper, we introduce a methodology for the simultaneous determination of antioxidant and pro-oxidant activity on a single microplate in situ, assuming that the FRAP method can measure both antioxidant and pro-oxidant activity due to the generation of pro-oxidant Fe2+ ions in the Fenton reaction. Systematic research using this rapid screening method may help to distinguish between compounds with dominant antioxidant efficacy and those with dominant pro-oxidant effects. Our preliminary study has revealed a dominant pro-oxidant effect for compounds with a higher number of oxygen heteroatoms, especially sp2 hybridized compounds (such as those containing keto groups), such as flavonoids and plant extracts rich in these structural types. Conversely, catechins, carotenoids, and surprisingly, extracts from birch leaves and chestnut leaves have demonstrated dominant antioxidant activity over pro-oxidant. These initial findings have sparked significant interest in the systematic evaluation of a more extensive collection of compounds and plant extracts using the developed method.

Antibacterial Potential of Microwave-Assisted Extraction Prepared Hydrolates from Different Salvia Species.

Salvia is a widely used herb that also contains essential oils and other valuable compounds. In this work, the hydrolates of five Salvia sp. were evaluated for their potential antimicrobial and antioxidant activity against four bacterial strains. The hydrolates were obtained from fresh leaves by microwave-assisted extraction. Chemical composition analysis by gas chromatography and mass spectrometry revealed that their major constituents were isopulegol (38.2-57.1%), 1,8-cineole (4.7-19.6%), and thujone (5.6-14.1%). The minimum inhibitory concentration (MIC) of the plant hydrolates was tested by the microdilution method at concentrations ranging from 1.0 to 512 µg/mL. The hydrolates prepared from Salvia officinalis and S. sclarea showed inhibitory activity on the tested Gram-positive and Gram-negative bacteria, taxon Salvia nemorosa showed inhibitory activity only partially. The hydrolate of S. divinorum had practically no antibacterial effect. Enterobacter asburiae was the only bacterium for which we found sensitivity to the hydrolate of S. aethiopis, with a MIC50 value of 216.59 µL/mL. The antioxidant activity of the hydrolates was low, ranging from 6.4 to 23.3%. Therefore, salvia hydrolates could be used as antimicrobial agents in medicine, cosmetics, and food preservation.

Insights into the early stage of Pinus nigra Arn. somatic embryogenesis using discovery proteomics.

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Arn. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryogenic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing. BIOLOGICAL SIGNIFICANCE: Somatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Arn. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues.

Using 7 cm immobilized pH gradient strips to determine levels of clinically relevant proteins in wheat grain extracts.

The aim of the work was to test a relatively simple proteomics approach based on phenol extraction and two-dimensional gel electrophoresis (2-DE) with 7 cm immobilized pH gradient strips for the determination of clinically relevant proteins in wheat grain. Using this approach, 157 2-DE spots were quantified in biological triplicate, out of which 55 were identified by matrix-assisted laser desorption/ionization - time of flight tandem mass spectrometry. Clinically relevant proteins associated with celiac disease, wheat dependent exercise induced anaphylaxis, baker's asthma, and food allergy, were detected in 24 2-DE spots. However, alcohol-soluble gliadins were not detected with this approach. The comparison with a recent quantitative study suggested that gel-based and gel-free proteomics approaches are complementary for the detection and quantification of clinically relevant proteins in wheat grain.

Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses.

Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map. Proteins were isolated from fully developed mature leaves in biological triplicate and analyzed by two-dimensional electrophoresis plus tandem mass spectrometry. Using this approach, we were able to reproducibly quantify 190 abundant protein spots, from which 157 proteins were identified. Most of identified proteins are associated with the energy and protein destination/storage categories. The reference map provides a basis for understanding the accumulation of flavonoids and other phenolic compounds in mature leaves (e.g., identification of chalcone synthase, the first committed enzyme in flavonoid biosynthesis). We additionally detected several proteins of as yet unknown function. These proteins comprise a pool of potential targets that might be useful in nontraditional medical applications.

Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency.

Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In this study, maize immature zygotic embryos were used to generate embryogenic (EC) and non-embryogenic (NEC) calli. In order to compare proteomes of EC and NEC, two-dimensional electrophoresis (2-DE) in combination with mass spectrometry was used. This approach resulted into 361 quantified 2-DE spots out of which 44 were found statistically significantly differentially abundant between EC and NEC. Mass spectrometry provided the identity for 23 proteins that were classified into 8 metabolic categories. The most abundant were proteins associated with energy followed by proteins associated with disease and defense. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. BIOLOGICAL SIGNIFICANCE: Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In order to further advance understanding of this biological phenomenon, proteomes of embryogenic and non-embryogenic callus, derived from immature zygotic embryos of inbred maize line A19, were compared using 2-DE based proteomic technology. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. This article is part of a Special Issue entitled: Environmental and structural proteomics.

MS(E) based multiplex protein analysis quantified important allergenic proteins and detected relevant peptides carrying known epitopes in wheat grain extracts.

The amount of clinically relevant, allergy-related proteins in wheat grain is still largely unknown. The application of proteomics may create a platform not only for identification and characterization, but also for quantitation of these proteins. The aim of this study was to evaluate the data-independent quantitative mass spectrometry (MS(E)) approach in combination with 76 wheat allergenic sequences downloaded from the AllergenOnline database ( www.allergenonline.org ) as a starting point. Alcohol soluble extracts of gliadin and glutenin proteins were analyzed. This approach has resulted in identification and quantification of 15 allergenic protein isoforms that belong to amylase/trypsin inhibitors, γ-gliadins, and high or low molecular weight glutenins. Additionally, several peptides carrying four previously discovered epitopes of γ-gliadin B precursor have been detected. These data were validated against the UniProt database, which contained 11764 Triticeae protein sequences. The identified allergens are discussed in relation to Baker's asthma, food allergy, wheat dependent exercise induced anaphylaxis, atopic dermatitis, and celiac disease (i.e., gluten-sensitive enteropathy). In summary, the results showed that the MS(E) approach is suitable for quantitative analysis and allergens profiling in wheat varieties and/or other food matrices.

The MS(E)-proteomic analysis of gliadins and glutenins in wheat grain identifies and quantifies proteins associated with celiac disease and baker's asthma.

Precise content of gliadin (Glia) and glutenin (Glu) proteins in wheat grain are largely unknown despite their association with celiac disease, various allergies, and physical processing properties of wheat. Developing methods to quantitatively measure clinically relevant proteins could support advancement in understanding exposure thresholds and clinical study design. The aim of this study was to use a data-independent mass spectrometry (MS(E)) approach for quantifying gliadin and glutenin proteins in wheat grain. The biologically replicated analysis yielded concentrations for 34 gliadin and 22 glutenin proteins. The primary focus of this survey was on measuring celiac disease proteins and baker's asthma associated proteins along with the proteins associated with viscoelastic properties of wheat flour and grain texture. The technical coefficients of variation ranged from 0.12 to 1.39 and indicate that MS(E) proteomics is a reproducible quantitative method for the determination of gliadin and glutenin content in the highly complex matrix of protein extracts from wheat grain. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Soybeans grown in the Chernobyl area produce fertile seeds that have increased heavy metal resistance and modified carbon metabolism.

Plants grow and reproduce in the radioactive Chernobyl area, however there has been no comprehensive characterization of these activities. Herein we report that life in this radioactive environment has led to alteration of the developing soybean seed proteome in a specific way that resulted in the production of fertile seeds with low levels of oil and ß-conglycinin seed storage proteins. Soybean seeds were harvested at four, five, and six weeks after flowering, and at maturity from plants grown in either non-radioactive or radioactive plots in the Chernobyl area. The abundance of 211 proteins was determined. The results confirmed previous data indicating that alterations in the proteome include adaptation to heavy metal stress and mobilization of seed storage proteins. The results also suggest that there have been adjustments to carbon metabolism in the cytoplasm and plastids, increased activity of the tricarboxylic acid cycle, and decreased condensation of malonyl-acyl carrier protein during fatty acid biosynthesis.

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes.

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.

Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings.

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.