Hormonal and antioxidant status of Physcia stellaris (L.) Nyl. populations growing in different natural zones of the Republic of Bashkortostan

Nowadays, the question concerning the role of lichens as indicators of the state of the environment remains urgent. The main aim of our research was to elucidate how different external factors influence their habitat and to establish the effects of anthropogenic load on morphological and physiological characteristics of lichen thallus. Mechanisms regulating growth responses of lichens are scarcely discussed and, therefore, it seemed interesting to study a possible role of the hormonal system in the formation of morphological characteristics depending on climate and ecological peculiarities of the lichen habitat. We collected Physcia stellaris (L.) Nyl. lichen in forests of the forest-steppe (Alsheevsky district, 53°58'06.5''N, 55°03'7.56''E), mountain forest (Ishimbaysky district, 53°62'50''N, 56°60'7.9''E) zones and in the parks of Sterlitamak (53°62'9,9''N, 55°9'3,01''E) of the Republic of Bashkortostan. In the species P. stellaris, we examined morphological and physiological features of the thallus: sizes of algal and fungal hyphae cells, biomass, length, area, number of blades and apothecia, enzyme activity and hormonal status. To compare the extent of shading in lichen habitats, we measured illumination with the help of luxmeter at the clearing in the woods and under the trees. Air humidity was measured with a humidity controller. To determine physiological and biochemical characteristics, we carried out lichen rehydration, when hormonal system and enzymes were recovered. To achieve this, lichens were placed in a moist camera for 14 h at 10±2°С and 80-90% relative air humidity. Phytohormones were determined after their extraction, purification and concentration with the help of solid phase enzyme immunoassay. To determine the activity of enzymes, pre-incubation of dry samples was carried out and then samples were homogenized and centrifuged twice at 10000 rotations per minute at 4°С. Catalase activity was determined according to the remaining quantity of hydorogen peroxide forming a complex with ammonium molybdate. To determine the sizes of fungus hyphae cells, temporary preparations were conducted. 0.5 g of preliminary moistened lichen samples was grinded in 5 ml of water and the obtained homogenate was observed under light microscope Axio Imager (Carl Zeiss, Jena, Germany) at different magnifications with the help of digital camera AxioCam MRc 5 (Carl Zeiss, Jena, Germany). Not less than 50 cells of algae and fungi were measured in each temporary preparation and algae cell volume was calculated according to sphere formula V=1/6nd³, where d is cell diameter. The volume of fungus hyphae was calculated according to ellipsoid formula: V=4/3nabc=4/3nab², where a is half of the cell length, while b is half of the width of cells (the values of radiuses b and c are assumed to be equal). We selected samples at virginal and generative stages of ontogenesis from the spectrum of population for cytological studies. Individuals at early stages were not considered. X-ray fluorescent analysis of SO₃ content in dried lichen samples was performed with an energy dispersive X-ray fluorescence spectrometer of the EDX type (Schimadzu, Japan). Thallomes of P. stellaris lichen from Sterlitamak had smaller biomass, area, average length, number of blades and apothecies, compared to samples growing in Ishimbaysky and Alscheevsky districts (See Table 1). There were no significant differences between the samples of lichens grown in Ishimbaysky and Alsheevsky districts. Measurement of the size of the hyphae cells of fungi and algae of the lichen thallus showed that in Sterlitamak they were smaller than in Alscheyevsky and Ishimbaysky districts. We also established that in the samples of lichens from Alsheevsky district, the size of the cells of fungi and algae was less than from Ishimbaysky district (See Table 2). About 40% higher content of sulfur oxide was detected in the samples collected in the city as compared to those from Ishimbaysky and Alsheevsky districts (See Table 1). A high activity of catalase enzyme was detected in samples from Sterlitamak indicating participation of the antioxidant system in the processes of adaptation to the conditions of anthropogenic impact (See Table 3). The determination of the content of phytohormones (IAA, ABA, cytokinins and the sum of zeatin derivatives) demonstrated different hormonal status in lichens growing in different habitat conditions (See Table 4). Samples collected in the city showed a high level of ABA and relatively low concentrations of auxins and cytokinins in lichen tissues. It is suggested that a relatively low growth activity of lichens collected in the city may be associated with the reaction of the hormonal system. The hormonal status of lichens growing in different natural zones was also shown to be different. Certain relationship was revealed between the high level of auxin content and the larger size of the photobiont cells in Ishimbaysky district. On the contrary, smaller cells of a photobiont in Alsheevsky district were observed against a background of a high level of cytokinins in the tissues of lichens. Thus, estimation of only external growth indexes was insufficient for using lichen as indicators. Cell sizes, the content of pollutants in the tissues and the state of antioxidant and hormonal systems were also found to be informative.

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