Stanisław Weidner

Alterations in root proteome of salt-sensitive and tolerant barley lines under salt stress conditions

Salinity is one of the most important abiotic stresses causing a significant reduction of crop plants yield. To gain a better understanding of salinity tolerance mechanisms in barley (Hordeum vulgare), we investigated the changes in root proteome of salt-sensitive (DH14) and tolerant (DH187) lines in response to salt-stress. The seeds of both barley lines were germinating in water or in 100mM NaCl for 6 days. The root proteins were separated by two-dimensional gel electrophoresis. To identify proteins regulated in response to salt stress, MALDI-TOF/TOF mass spectrometry was applied. It was demonstrated that the sensitive and tolerant barley lines respond differently to salt stress. Some of the identified proteins are well-documented as markers of salinity resistance, but several proteins have not been detected in response to salt stress earlier, although they are known to be associated with other abiotic stresses. The most significant differences concerned the proteins that are involved in signal transduction (annexin, translationally-controlled tumor protein homolog, lipoxygenases), detoxification (osmotin, vacuolar ATP-ase), protein folding processes (protein disulfide isomerase) and cell wall metabolism (UDP-glucuronic acid decarboxylase, β-d-glucan exohydrolase, UDP-glucose pyrophosphorylase). The results suggest that the enhanced salinity tolerance of DH187 line results mainly from an increased activity of signal transduction mechanisms eventually leading to the accumulation of stress protective proteins and cell wall structure changes.

Extracts of Phenolic Compounds from Seeds of Three Wild Grapevines—Comparison of Their Antioxidant Activities and the Content of Phenolic Compounds

Phenolic compounds were extracted from three wild grapevine species: Vitis californica, V. riparia and V. amurensis seeds using 80% methanol or 80% acetone. The total content of phenolic compounds was determined utilizing the Folin-Ciocalteu’s phenol reagent while the content of tannins was assayed with the vanillin and BSA precipitation methods. Additionally, the DPPH free radical scavenging activity and the reduction power of the extracts were measured. The RP-HPLC method was applied to identify the phenolic compounds in the extracts, such as phenolic acids and catechins. The seeds contained large amounts of tannins, catechins and gallic acid and observable quantities of p-coumaric acid. The total content of phenolic compounds and tannins was similar in the extracts from V. californica and V. riparia seeds. However, the total content of total phenolic compounds and tannins in the extracts from V. californica and V. riperia seeds were about two-fold higher than that in the extracts from V. amurensis seeds. Extracts from seeds of the American species (V. californica and V. riparia) contained similarly high concentrations of tannins, whereas extracts from seeds of V. amurensis had approximately half that amount of these compounds. The content of catechin and epicatechin was similar in all extracts. The highest DPPH• anti-radical scavenging activity was observed in the acetonic and methanolic extracts of V. californica and V. riparia seeds— while the acetonic extract from the V. californica seeds was the strongest reducing agent.

Proteomic analysis of response to long-term continuous stress in roots of germinating soybean seeds

Germination is a complex process, highly dependent on various environmental factors, including temperature and water availability. Germinating soybean seeds are especially vulnerable to unfavorable environmental conditions and exposure to long-term abiotic stresses may result in diminishing much of the yield and most importantly - restrained germination. In the present study, a proteomic approach was employed to analyze influence of cold and osmotic stress on roots of germinated soybean (Glycine max, L.) seeds. Seeds were germinating under continuous conditions of cold stress (+10°C/H2O), osmotic stress (+25°C/-0.2MPa) as well as cold and osmotic stress combined (+10°C/-0.2MPa). Proteome maps established for control samples and stress-treated samples displayed 1272 CBB-stained spots. A total of 59 proteins, present in both control and stress-treated samples and showing significant differences in volume, were identified with LC/nanoESI-MS. Identified proteins divided into functional categories, revealed 9 proteins involved in plant defense, 8 proteins responsible for plant destination and storage and 10 proteins involved in various tracks of carbohydrate metabolism. Furthermore, a number of proteins were assigned to electron transport, range of metabolic pathways, secondary metabolism, protein synthesis, embryogenesis and development, signal transduction, cellular transport, translocation and storage. By analyzing differences in expression patterns, it was possible to trace the soybean response to long-term abiotic stress as well as to distinguish similarities and differences between response to cold and osmotic stress.

Proteomic changes in the roots of germinating Phaseolus vulgaris seeds in response to chilling stress and post-stress recovery.

Plants respond to different environmental cues in a complex way, entailing changes at the cellular and physiological levels. An important step to understand the molecular foundation of stress response in plants is the analysis of stress-responsive proteins. In this work we attempted to investigate and compare changes in the abundance of proteins in the roots of bean (Phaseolus vulgaris L.) germinating under long continuous chilling conditions (10°C, 16 days), exposed to short rapid chilling during germination (10°C, 24h), as well as subjected to recovery from stress (25°C, 24h). The results we obtained indicate that germination under continuous chilling causes alterations in the accumulation of the proteins involved in stress response, energy production, translation, vesicle transport, secondary metabolism and protein degradation. The subsequent recovery influences the accumulation of the proteins implicated in calcium-dependent signal transduction pathways, secondary metabolism and those promoting cell division and expansion. Subjecting the germinating bean seeds to short rapid chilling stress resulted in a transient changes in the relative content of the proteins taking part in energy production, DNA repair, RNA processing and translation. Short stress triggers also the mechanisms of protection against oxidative stress and promotes expression of anti-stress proteins. Subjecting bean seeds to the subsequent recovery influences the abundance of the proteins involved in energy metabolism, protection against stress and production of phytohormones. The exposure to long and short chilling did not result in the alterations of any proteins common to both treatments. The same situation was observed with respect to the recovery after stresses. Bean response to chilling is therefore strongly correlated with the manner and length of exposure to low temperature, which causes divergent proteomic alterations in the roots.

BIOLOGICAL ACTIVITY OF GRAPEVINE PHENOLIC COMPOUNDS

Phenolic compounds present in nearly all parts of grape berries are in-creasingly believed to exhibit antioxidant and antimicrobial activities and to play a significant role in the prevention of diseases including cancer and cardio-vascular diseases (Bagchi et al. 2000, Ariga 2004). The majority of studies on grape phenolics properties has been conducted using proanthocyanidin-rich seeds extracts (GSEs). Grape seed extract is rich in phenolic acids and different flavonoids but most important are the monomeric flavan-3-ols [(+)-catechin, (-)-epicatechin, and (-)-epicatechin-3-

Metabolism of phenolic compounds in Vitis riparia seeds during stratification and during germination under optimal and low temperature stress conditions

We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.

Phenolic compounds in embryos of triticale caryopses at different stages of development and maturation in normal environment and after dehydration treatment

High performance liquid chromatography analysis revealed the presence of five phenolic acids in embryos of triticale caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic). Free phenolic acids reached the highest level at the early stages of development when germination was the lowest and decreased considerably in embryos at the final stage of grain maturation when germination was the highest. Revealing inverse correlation between the contents of free phenolic acids in developing embryos and intensity of precocious germination may indicate a role of phenolic acids in preventing pre-harvest sprouting in cereals. Total content of phenolic acids in embryos (i.e. free, and liberated from soluble esters and glycosides) increased gradually to 43DAF and decreased at full ripeness, whereas the content of total phenolics fluctuated slightly in embryos during development and ripening of caryopses. Enforced dehydration of unripe triticale caryopses stimulated germination both in embryos and whole grains. During the enforced dehydration treatment, a decrease in total content of both phenolic acids and phenolic compounds in embryos of triticale caryopses at different stages of development and maturation was observed. It should be stressed, that a number of naturally occurring phenolics are known to inhibit the germination of cereal grains. A possible role for phenolics in preventing pre-harvest sprouting and acclimation to dehydration in cereals is discussed.

Differences in the Phenolic Composition and Antioxidant Properties between Vitis coignetiae and Vitis vinifera Seeds Extracts

Phenolic compounds were extracted from European and Japanese grapevine species (Vitis vinifera and V. coignetiae) seeds using 80% methanol or 80% acetone. The total content of phenolic compounds was determined utilizing Folin-Ciocalteu’s phenol reagent, while the content of tannins was assayed by the vanillin and BSA precipitation methods. Additionally, the DPPH free radical and ABTS cation radical scavenging activities and the reduction power of the extracts were measured. The HPLC method was applied to determine the phenolic compounds, such as phenolic acids and catechins. The seeds contained large amounts of tannins and gallic acid and observable quantities of catechins, p-coumaric, ferulic and caffeic acids. The dominant form of phenolic acids in the extracts was the ester-bound form. The content of total phenolics was higher in the European grape V. vinifera seeds, which also contained more tannins, catechins and phenolic acids, except for caffeic acid. Extracts from V. vinifera seeds showed better radical scavenger properties and stronger reducing power. The total contents of phenolic compounds and tannins in acetone extracts were higher than in methanolic extracts. Acetone extracts also exhibited stronger antiradical properties as well as stronger reducing power.

Changes in the protein patterns in pea (Pisum sativum L.) roots under the influence of long- and short-term chilling stress and post-stress recovery

Amongst many factors restricting geographical distribution of plants and crop productivity, low temperature is one of the most important. To gain better understanding of the molecular response of germinating pea (Pisum sativum L.) to low temperature, we investigated the influence of long and short chilling stress as well as post-stress recovery on the alterations in the root proteomes. The impact of long stress was examined on the pea seeds germinating in the continuous chilling conditions of 10 °C for 8 days (LS). To examine the impact of short stress, pea seeds germinating for 72 h in the optimal temperature of 20 °C were subjected to 24-h chilling (SS). Additionally, both stress treatments were followed by 24 h of recovery in the optimal conditions (accordingly LSR and SR). Using the 2D gel electrophoresis and MALDI-TOF MS protein identification, it was revealed, that most of the proteins undergoing regulation under the applied conditions were implicated in metabolism, protection against stress, cell cycle regulation, cell structure maintenance and hormone synthesis, which altogether may influence root growth and development in the early stages of plant life. The obtained results have shown that most of detected alterations in the proteome patterns of pea roots are dependent on stress duration. However, there are some analogical response pathways which are triggered regardless of stress length. The functions of proteins which accumulation has been changed by chilling stress and post-stress recovery are discussed here in relation to their impact on pea roots development.

The effects of cold stress on the phenolic compounds and antioxidant capacity of grapevine (Vitis vinifera L.) leaves

According to some estimates, a 70% increase in crop yield could be achieved if the environmental conditions were close to the optimum ones for a given plant, which is why the identification and control of adverse environmental effects is a top priority in many countries worldwide. This paper contains a discussion of the changes in selected elements of the secondary metabolism in the leaves of two grapevine varieties (Vitis vinifera L.) with a different degree of tolerance to cold stress during prolonged and constant low temperature stress. The analyses have shown that the more-tolerant variety was characterized by a higher content of phenolic compounds, better radical-scavenging capacity and stronger reducing power. However, the cold stress caused a decrease in the concentration of the phenolics and decreased the scavenging capacity in the leaves of both varieties. Four phenolic acids have been identified in the extracts from the leaves of both grapevines: caffeic acid, p-coumaric acid, ferulic acid and a caffeic acid derivative. Caffeic acid appeared in the highest concentrations in all the leaf extracts. Additionally, it has been noted that in the leaves of the varieties susceptible and tolerant to cold stress, the prolonged exposure to low temperature caused a considerable reduction of the content of all identified phenolic acids. The results of the analyses have demonstrated large differences in the functioning of the secondary metabolism in response to the same stressor.