Environmental impacts on intraspecific variation in Ambrosia artemisiifolia genome size in Slovakia, Central Europe.

The quantity of DNA in angiosperms exhibits variation attributed to many external influences, such as environmental factors, geographical features, or stress factors, which exert constant selection pressure on organisms. Since invasive species possess adaptive capabilities to acclimate to novel environmental conditions, ragweed (Ambrosia artemisiifolia L.) was chosen as a subject for investigating their influence on genome size variation. Slovakia has diverse climatic conditions, suitable for testing the hypothesis that air temperature and precipitation, the main limiting factors of ragweed occurrence, would also have an impact on its genome size. Our results using flow cytometry confirmed this hypothesis and also found a significant association with geographical features such as latitude, altitude, and longitude. We can conclude that plants growing in colder environments farther from oceanic influences exhibit smaller DNA amounts, while optimal growth conditions result in a greater variability in genome size, reflecting the diminished effect of selection pressure.

Endosidin 2 accelerates PIN2 endocytosis and disturbs intracellular trafficking of PIN2, PIN3, and PIN4 but not of SYT1.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.

Genotoxic assessment of selected native plants to deferentially exposed urban ecosystems.

The aim of this study is to provide an analysis of the impact of ecogenotoxicity on native flora abortivity in various urban areas. In which, there was an analysis of 5 groupings of locations with a differing environmental load intensity within the city of Bratislava (Slovakia) over a 2-year period. Our results show varying data depending on the proximity of each site relating to a direct source of pollution and the potential impact of localized wind currents on the distribution of pollutants in the urban environment. The highest value of pollen abortivity in the city was observed in a group of locations exposed to heavy traffic pollution loads. Abortivity of native flora near heavy traffic road areas correlated with the imissions data measured in the same area. Wind-exposed uncovered sites also experienced higher values of native flora abortivity. These results confirmed the varying intensity of genotoxic impact in differing localities and also suggest that xenobiotic effects on flora can occur remotely from the original source of pollution.

The ecogenotoxic plant biomonitoring of a long-term polluted area in central Slovakia.

The aim of the study was to analyze, update, and complete the results of research in the field of in situ phytoindication of environmental genotoxicity near the aluminum plant in Ziar nad Hronom in central Slovakia. The authors focused on two methodologies: pollen abortivity assay of native flora and Trad-MCN assay. Comparison of changes in responses of living systems to changes in precipitation was conducted, and it suggests that there is an existence of an impact from a dilution effect to the plants. Also, the gradual increase of pollen abortivity in the 1990s and its decrease from 2009 were observed in a majority of species of wild flora. On an annual basis, abortivity has declined, although it has risen up slightly within each season. Despite a gradual decrease in the micronucleus frequency, the study area is still influenced by the ecogenotoxic factors. Pollen analysis of native flora and introduced Tradescantia plants indicates the long-time presence of ecogenotoxicity in this region due to the presence of aluminum plant.

The Tradescantia micronucleus assay is a highly sensitive tool for the detection of low levels of radioactivity in environmental samples.

Environmental contamination with radioactive materials of geogenic and anthropogenic origin is a global problem. A variety of mutagenicity test procedures has been developed which enable the detection of DNA damage caused by ionizing radiation which plays a key role in the adverse effects caused by radioisotopes. In the present study, we investigated the usefulness of the Tradescantia micronucleus test (the most widely used plant based genotoxicity bioassay) for the detection of genetic damage caused by environmental samples and a human artifact (ceramic plate) which contained radioactive elements. We compared the results obtained with different exposure protocols and found that direct exposure of the inflorescences is more sensitive and that the number of micronuclei can be further increased under "wet" conditions. The lowest dose rate which caused a significant effect was 1.2 µGy/h (10 h). Comparisons with the results obtained with other systems (i.e. with mitotic cells of higher plants, molluscs, insects, fish and human lymphocytes) show that the Tradescantia MN assay is one to three orders of magnitude more sensitive as other models, which are currently available. Taken together, our findings indicate that this method is due to its high sensitivity a unique tool, which can be used for environmental biomonitoring in radiation polluted areas.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis.

By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.

Expansion and aerobiology of Ambrosia artemisiifolia L. in Slovakia.

INTRODUCTION AND OBJECTIVE: The invasive alien species Ambrosia artemisiifolia cause environmental, agronomical and medical problems in many regions of the world, including Slovakia. The purpose of this study was to survey the spread and distribution of this species in Slovakia and to analyse its airborne pollen pattern. MATERIALS AND METHODS: To evaluate the spatiotemporal dynamics of Ambrosia invasion in the territory of Slovakia, herbarium specimens, published databases and field investigations were considered. Aerobiological sampling was based on the analysis of pollen records at five aerobiological stations in Slovakia. For Bratislava and Banská Bystrica Monitoring stations, trends in Ambrosia pollen seasons were determined using Mann-Kendall test and Sen's slope estimator. RESULTS: Since the first record of A. artemisiifolia in Slovakia, the number of its colonies and its spread rate has increased considerably, and the colonisation of this species has been successful mainly in the south-western part of the country. Highest airborne pollen counts were recorded in Nitra, Trnava and Bratislava Monitoring Stations situated in the areas most infested by A. artemisiifolia in Slovakia. However, high pollen counts were also noted in Banská Bystrica and Kosice Monitoring Stations situated in areas where the source species was less abundant. During the study period, the number of days on which the pollen concentration exceeded the threshold of sensitivity increased significantly (+1.33 days/year) in Banská Bystrica, whereas the peak value decreased significantly (-13.37 pollen/year) in Bratislava. CONCLUSION: The number of the populations of A. artemisiifolia has increased considerably in recent years. Besides the most infested areas, high airborne pollen counts were also recorded in territories where the plant species was less abundant. During the study period, the intensity of Ambrosia pollen seasons decreased in Bratislava, probably due to changes in land-use practices, while the increasing trend in the pollen seasons intensity in Banská Bystrica mainly reflects the situation in the ragweed-infested remote areas due to long-range pollen transport.

Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

Influence of airborne pollen counts and length of pollen season of selected allergenic plants on the concentration of sIgE antibodies on the population of Bratislava, Slovakia.

INTRODUCTION AND OBJECTIVE: The association between airborne pollen counts or duration of pollen season and allergy symptoms is not always distinguished. The purpose of this study was to examine the correlation between pollen exposure (annual total pollen quantity and main pollen season length) of selected allergenic plants in the atmosphere of Bratislava, and concentration of allergen-specific immunoglobulin E (sIgE) in serum of patients with seasonal allergy during 2002-2003. MATERIALS AND METHODS: The concentration of pollen was monitored by a Burkard volumetric pollen trap. At the same time, 198 pollen allergic patients were testing to determine the values of sIgE antibodies against selected pollen allergens; a panel of 8 purified allergens was used. RESULTS: The highest percentages of sensitization were detected for Poaceae and Ambrosia pollen allergens. The most abundant airborne pollen types were Urticaceae, Betula, Populus, Fraxinus, Pinus and Poaceae. The length of the pollen season varied. The longest pollen season was that of the Plantago - 105 days, and the shortest, Corylus - 20 days. A significant correlation was found between annual total pollen quantity and median sIgE values, especially in 2002. CONCLUSIONS: A strong and significant positive correlation was observed between pollen counts, excluding Betula, and sIgE levels in both analysed years. The correlation was weaker and negative in the case of length of pollen season and sIgE values.

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion.

Arabidopsis synaptotagmin 2 (SYT2) has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen of Arabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmMan1-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

Ageing of trees: application of general ageing theories.

The main questions posed in ageing theories are how ageing evolved and whether or not it is programmed. While these questions have not yet been clearly resolved, several groups of possible theories have been published on this topic. However, most of these theories do not consider plants, and the specific traits involved in their ageing mechanisms. The first trait covers clonality and sectoriality and the second concerns the lack of a differentiated germ line. The lack of a germ line prevents telomere shortening which can lead to the transfer of somatic mutations into sexual offspring, while sectoriality in trees causes isolation of potentially catastrophic events in one tree part, thus creating a population of more or less independent modules within one axis. The processes of population dynamics, including ageing, can act within the framework of an individual tree as well as in that of the population as a whole, although the processes involved differ and consequently result in different effects.

Study of gherkin lactase in cell culture and in seedlings.

A synthetic substrate replacing lactose has facilitated application of a simple, rapid and sensitive method for the identification and determination of extracellular and intracellular gherkin lactase. The intracellular enzyme activity was estimated from the cell suspension, while the extracellular enzyme activity was established within the cell free cultivation medium. A suspension of gherkin cells was permeabilized by Tween 20, or Tween 80, or hexadecyltrimethyl ammonium bromide, or hexadecylpyridinium chloride or ethanol added one at a time and then immobilized by glutaraldehyde. The highest lactase activity was at pH 4.8 at a temperature of 55°C. The hydrolysis of substrate was linear for 4.5h and reached 60% conversion. The cells had high lactase activity and good stability. During long-term storage they demonstrated convenient physico-mechanical properties.

Wild plant species in bio-indication of radioactive-contaminated sites around Jaslovské Bohunice nuclear power plant in the Slovak Republic.

In a long-term study of contaminated soil around Jaslovské Bohunice nuclear power plant (NPP), 24 species of local flora were used to show impact of serious accidents from 1976 to 1977. The 19-km-long banks of the Jaslovské Bohunice NPP wastewater recipient have been identified as contaminated by (137)Cs. In total, more than 67,000m(2) of riverbanks have been found as being contaminated at levels exceeding 1Bq (137)Csg(-1) of soil. Used phytotoxic and cytogenetic "in situ" tests were extended by analyses of pollen grains. Although the dose of some samples of radioactive soil was relatively high (322kBqkg(-1)) no significant impact on the biological level of tested wild plant species was observed.

In situ biomonitoring of the genotoxic effects of mixed industrial emissions using the Tradescantia micronucleus and pollen abortion tests with wild life plants: demonstration of the efficacy of emission controls in an eastern European city.

Aim of the study was to monitor changes of genotoxic activity of urban air caused by an incinerator and a petrochemical plant in Tradescantia micronucleus (Trad-MCN) and pollen fertility assays with wild plants (Chelidonium majus, Clematis vitalba, Cichorium intybus, Linaria vulgaris, Robinia pseudoacacia). While in the first sampling period (1997-2000) significantly (on average 80%) more MN were found at the polluted site in comparison to controls from a rural area, no significant effects were observed during a later period (between 2003 and 2005). A similar pattern was observed in the pollen abortion assays in which the most pronounced effects were found in chicory and false acacia. The differences of the results obtained in the two periods can be explained by a substantial reduction of air pollution by use of new technologies. In particular the decrease of SO(2) emissions may account for the effects seen in the present study.

Bioassay for the determination of the intra- and extracellular activity of aminopeptidases in immobilized tomato cells.

Permeabilized tomato cells were cross-linked with glutaraldehyde in the absence of a carrier. The immobilized cells demonstrated significantly lower aminopeptidase (AP) activities than untreated control cells. However, when immobilized with pectate and alginate gels, the tomato cells retained their AP activities. A new method for the determination of the activity of both extra- and intracellular AP was developed, based on enzyme-catalyzed hydrolysis of a series of synthetic beta-naphthylamides (betaNA) of the L-amino acids Ala, Arg, Leu, Pro, Tyr, or of the synthetic beta-methoxynaphthylamides (betaMNA) of Ala and Arg. Extracellular AP--produced by calli, cell-suspension culture, or seedlings of tomato cells grown on agar--hydrolyzed these peptidic substrates to the free naphthalene amines and amino acids. Staining with Fast Garnet GBC salt under formation of bright reddish azo dyes readily allowed the determination of AP activities. For the tomato-cell suspension, the intracellular activity accounted for 91.3-93.9% of the total activity, and the extracellular one for 6.1-8.7%, respectively. Our method permits the rapid, simple, and specific determination of plant aminopeptidases.

Study of immobilized and extracellular saccharase of watermelon.

A simple, rapid and reproducible procedure for the identification and determination of extracellular saccharase from culture medium of watermelon cell suspension cultures is described. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. Watermelon cell suspension was permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest saccharase activity was at pH 4.6 at a temperature of 50 degrees C. The hydrolysis of substrate was linear 5h after reaching 60% conversion. The cells had high saccharase activity and good stability, and in long-term storage they showed convenient physico-mechanical properties.

In situ monitoring of clastogenicity of ambient air in Bratislava, Slovakia using the Tradescantia micronucleus assay and pollen abortion assays.

Aim of this study was to monitor the genotoxic effects of polluted air in Bratislava (Slovakia) with the Tradescantia micronucleus (Trad-MN) test. In situ monitoring was carried out at five locations during two seasons (years 2003 and 2004). Flower pots with Tradescantia paludosa (clone 03) plants were exposed for 6-8 weeks at the different sites each year. The highest MN levels were observed in the vicinity of an agrochemical factory (3.1 times higher than background level in 2003 and 2.7 times higher in 2004). Lower effects were seen when plants were exposed to urban traffic emissions or in the vicinity of a glass-producing plant (the MN frequencies ranged between 2.8 and 4.4 per 100 tetrads, respectively, while the control frequencies were 2.1-2.6 per 100 tetrads); exposure near a petrochemical plant had no significant effects. In pollen abortion assays, three wild growing species were used, namely, chicory (Cichorium intybus L.), old man's beard (Clematis vitalba L.) and common toadflax (Linaria vulgaris Mill.). Again, the strongest effects were observed close to the agrochemical industry (reduction of fertile pollen by 5.6%, 11.1% and 8.3% in chicory, old mans beard and in toadflax, respectively). Cichorium intybus was the most sensitive species and the number of abortive pollen grains was 5.1 times higher in specimens collected near the agrochemical factory than that seen at the control location. These observations indicate that contaminated urban air has an impact on the fertility of wild plants. Furthermore, it is interesting that the same rank order of effects was seen in pollen abortion assays as in the Trad-MN test (agrochemical industry>technical glass industry≥traffic>city incinerator/petrochemical plant). These results confirm the sensitivity of the Tradescantia MN test and pollen abortion assays for the detection of air pollution, and show that distinct differences exist in genotoxicity of different sources of pollutants.

Plant bioassays for an in situ monitoring of air near an industrial area and a municipal solid waste: Zilina (Slovakia).

The process of a bioindication of genotoxic effects of complex mixtures on the environment using higher plants is very appropriate and effective. We present the results of an in situ indication of the genotoxic effects of polluted environment near Zilina city. For a more complex monitoring we used: the Tradescantia micronucleus (Trad-MCN) assay, the Tradescantia microspore test and an evaluation of the abortivity of the pollen grains of native plant species. We found significant differences in the frequency of the micronuclei when using the Trad-MCN test in local of Duben. The Tradescantia pollen abortivity test showed significant differences in the frequency of the abortive pollen grains between the exposed groups and the control group. By using native plant species in the pollen abortivity test we found significant differences in both of the two locations for the four following species during two consecutive years: Artemisia vulgaris, Melilotus albus, Trifolium pratense, Typha latifolia.

Localization and measurement of extracellular plant galactosidases.

A simple, rapid and sensitive procedure for the identification and determination of plant extracellular alpha-galactosidase and beta-galactosidase is described using callus cultures and seedlings from tomato. Synthetic substrates (1-naphthyl- and p-nitrophenyl-alpha-D- and beta-D-galactopyranosides) were used for the identification and determination of intracellular and extracellular activity of alpha-galactosidase and beta-galactosidase, respectively. Many iminosugars or azasugars are strong glycosidase inhibitors and some of them show promising chemotherapeutic effects against viral diseases, and are potentially antidiabetic agents, as well as antitumor agents. These facts initiated our interest in a rapid and sensitive assay to determine activity of alpha-galactosidase and beta-galactosidase in plant tissues. The results presented here show the potential of the assay of the activity of intracellular and extracellular galactosidases of plant origin in inhibitory and/or biotechnological studies.