Karol Mičieta

Three Species of Genus Pinus Suitable as Bioindicators of Polluted Environment

The long-term study objective was to determine the suitability of selected species of genus Pinus (Pinus sylvestris, Pinus nigra and Pinus mugo) for use as bioindicators of environmental pollution. In these experiments a new methodology of evaluation of mitotic abnormalities was established and its potential for common use tested. The study confirmed three species of genus Pinus as suitable bioindicators. The results also indicate that trees may show adaptive changes in response to severe pollution stress.

Microspore analysis for genotoxicity of a polluted environment

Abstract The advantage of field grown plants as bioindicators of a polluted environment is demonstrated through analyses of microspores. Forty-one species of the native Slovakian flora were used to test the hypothesis that local flora can serve as bioindicators of genotoxicity of industrially polluted areas. A higher frequency of aberrant microspores from plants in the polluted areas supported the hypothesis.

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.

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

Auxins and auxin transport inhibitors affect PIN-FORMED2 (PIN2) dynamics in Arabidopsis epidermal root cells through the synthetic and secretion pathway, but they do not inhibit 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.

Diurnal variation in airborne pollen concentration of six allergenic tree taxa and its relationship with meteorological parameters

In this study, we have evaluated 2-hourly variations of Betula, Carpinus, Populus, Fraxinus, Cupressaceae–Taxaceae and Pinus pollen concentrations in 24-h periods as well as the impact of weather variables on the diurnal patterns of the mentioned taxa. The study was conducted in Bratislava over a period of nine successive years (2002–2010). Based on the seasonally averaged diurnal patterns, the peak pollen values occurred mostly in the middle of the day between 12 a.m. and 14 p.m., while for Fraxinus, the peaks were denoted also in the afternoon or midnight for Betula and Cupressaceae–Taxaceae. Except for Pinus, the lowest pollen concentrations of all analysed taxa were recorded in the early morning hours between 02 a.m. and 06 a.m. The lowest Pinus pollen concentrations were observed at 22 p.m. We found significant positive relationships between airborne concentrations of pollen recorded at certain times of the 24-h period and air temperature, hours of sunshine and wind speed and a significant negative relationship with relative humidity.

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.

Tradescantia Micronucleus and Vicia Chromosome Anatelophase Assays in Monitoring of Genotoxicity of Urban Soil

Tradescantia micronucleus assay (Trad MCN assay) and Vicia anatelophase chromosome aberration assay (Vicia assay) were used to evaluate genotoxicity of extracts from soil samples. Soil samples from six different sites in area of Bratislava - capital of the Slovak Republic - have been taken. As a negative control soil samples from Botanical garden of medicinal plants, Faculty of Pharmacy, Comenius University were used. Soil samples for Trad MCN assay were extracted with distilled water or 5% dimethylsulphoxide (DMSO). Similarly genotoxicity of sample extracted with distilled water by Vicia assay was evaluated. Two sites were found as most polluted. The results of the study indicate different level of pollution in selected sites.

A Simple and Rapid Method of Identification of Extracellular Plant Galactosidases

A simple, rapid, and sensitive procedure for the identification of plant extracellular α- and β-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-6 day old seedlings of Californian poppy germinating on agar plates, and cell suspension cultures cultivated from callus cultures. Synthetic substrates were used for the determination of the intra- and extracellular activities of α- and β-galactosidase. Many imino- or azasugars are strong glycosidase inhibitors, and some of them show promising chemotherapeutic effects against viral disease and are potential antidiabetic as well as antitumor agents. This fact aroused our interest for a rapid and sensitive assay method of the activity of α- and β-galactosidases of plant origin. The results presented here show the perspective usefulness of extracellular galactosidases of plant origin in inhibitory and/or biotechnological studies.

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.

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.