Acta Physiol Plant (2007) 29 (Suppl 1):S13–S31 DOI 10.1007/s11738-007-0080-z © Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2007

oral presentations

The role of nitric oxide in the resistance of pelargonium leaves to Botrytis cinerea * M. Arasimowicz, * J. Floryszak-Wieczorek, ** G. Milczarek * Department of Plant Physiology, The August Cieszkowski Agricultural University, Wo³yñska 35, 60-637 Poznañ, Poland ** Institute of Chemistry and Technical Electrochemistry, Poznañ University of Technology, Piotrowo 3, 60-965 Poznañ, Poland Introduction: The primary object of interest was to explain the participation of nitric oxide (NO) in defense responses of the host plant induced by a necrotrophic pathogen - B. cinerea. Methods: Experiments were conducted on two cultivars of ivy-leaved pelargonium (Pelargonium peltatum L.), ‘Shiva’® – susceptible and ‘Cascade’® – resistant to Botrytis cinerea Pers. Two low-invasive techniques were used for in situ NO measurement, namely the electrochemical method using a microelectrode and the cytochemical method with DAF- 2DA. Moreover, H2O2 accumulation, the activity of CAT, APX, the antioxidant pool, ethylene production and PCD using the TUNEL assay in the host tissue were analyzed. Results: Pelargonium leaves in a response to B. cinerea showed NO production. In the resistant cultivar after a strong NO burst, the wave of secondary NO generation followed. The initial epicenter of NO synthesis was located in targeting cells which exhibited a TUNEL-positive reaction. Early after the challenge, the elevated level of H2O2 was correlated with the reversible inhibition of catalase and ascorbate peroxidase and the suppression of ethylene. Next induced NO generation was expanding and gradually disappearing in the successively provoked enhancement of the pool of antioxidants. In contrast, in the susceptible cultivar a weak NO burst was recorded and further NO generation increased only as the disease was progressing, which was accompanied by a huge H2O2 accumulation and ethylene synthesis. Also B. cinerea colonizing susceptible cells acquired the ability to produce considerable amounts of NO and enhanced nitrosative and oxidative stress in the host tissue. Conclusions: Early NO burst is a signal for the induction of non-specific resistance of pelargonium to a necrotrophic pathogen. The wave of secondary NO emission provoked non-cell death associated defense, following the rule that the concentration of NO is linked to its action. A lack of such metabolic regulation via NO in the susceptible genotype promotes disease development.

Leaf structure and proline accumulation under salt and water stress N.A. Belyavskaya, O.A. Chyzhykova, N.Yu. Kovtun, Yu.P. Sidnev, T.O. Palladina N.G. Kholodny Institute of Botany, Tereschenkivska str. 2, Kyiv, 01601, Ukraine Introduction: Salinity and water stress are major environmental constraints limiting crop yield in many regions of world. Plant growth and survival in unfavorable conditions is associated with protective mechanisms. This study examined the changes in leaf structure and role of proline as a protector against NaCl- or PEG-induced stress in maize seedlings. Methods: The seeds of Zea mays L. (hybrid Kolektyvnyi 225 MV) were soaked in distilled water during one day and then germinated and grown on Hoagland solution for 8 days. Salt stress conditions were made by exposure of 7-d-old seedlings to 0.1M NaCl or 0.1M PEG. The free proline content was determined spectrophotometrically. Conventional methods of light and electron microscopy were used.

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Results: Leaves with larger thickness of leaf lamina, mesophyll and lower epidermis height, relative volume of granae and stromal thylakoids in chloroplast as well as smaller relative volume of chloroplast stroma and plastoglobuli were found after salt stress compared to those in control. After PEG treatment, there were observed significant changes in many anatomical and ultrastructural variables, particularly in decrease in intercellular air space volume, chloroplast, plastoglobule and stromal thylakoid relative volumes as well as increase in relative volumes of vasculature and plastoglobuli. The water and salt stress conditions increased the proline contents in leaves per 33 % and 76 %, correspondingly. Conclusions: Results obtained at the tissue and cellular level on maize leaves after their treatments with NaCl and PEG are evidence of structural reorganization under stress conditions. It is suggested that enhanced proline accumulation at salt stress was caused by additional toxic influence of Na+ ions compared to PEG effect.

Signaling role of ROS and phytohormones in allelopathic interactions between plants R. Bogatek, K. Oracz, A. Gniazdowska Department of Plant Physiology, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland Allelopathy phenomenon describing a chemical interaction between plants is defined as direct or indirect harmful or beneficial effect of donor plant on recipient plant through the production of chemical compounds released into the environment. Those chemicals interfere with growth of neighboring plants by modifications of biochemical and physiological processes. Sunflower (Helianthus annuus L.) actively influences the growth of surrounding plants due to variety of phytotoxins identified in its tissue (Macias et al. 2002). We investigated mode of action of sunflower foliar extract during germination of mustard (Sinapis alba L.) seeds. Inhibition of mustard seed germination was associated with alterations in reserve mobilization and generation of energy in the catabolic phase of germination. (Kupid³owska et al. 2006). Additionally, sunflower allelopathic compounds induced oxidative stress manifested as enlarged production of reactive oxygen species (ROS) (Oracz et al. 2007). It correlated well with enhanced MDA level and electrolyte leakage (Bogatek et al., 2006). Therefore we suggested that in allelopathy stress ROS (H2O2) may act as signaling molecules leading to changes in hormonal balance during seed germination. ABA concentration in seeds increased after exposition to sunflower allelochemicals. In the contrast, ethylene emission by mustard seeds was strongly repressed (Gniazdowska et al. 2007). Low ethylene concentration resulted from inhibition of key enzyme activities of ethylene biosynthesis (ACS and ACO). The disturbances in phytohormones level lead to decreasing metabolic activity of the embryo resulting in inhibition of germination and growth of young seedlings. The relationship between ROS production and phytohormones in allelopathy interaction will be presented. References Bogatek R, Gniazdowska A, Zakrzewska W, Oracz K, Gawroñski SW. 2006. Allelopathic effects of sunflower extracts on mustard seed germination and seedling growth. Biol Plant 50:156-8. Gniazdowska A, Oracz K, Bogatek R. 2007. Phytotoxic effect of sunflower (Helianthus annuus L.) to hormonal balance (ABA : ethylene) in germinating mustard (Sinapis alba L.) seeds. Allelopathy J 19:215-26. Kupid³owska E, Gniazdowska A, Stêpieñ J, Corbineau F, Vinel D, Skoczowski A, Janeczko A, Bogatek R.2006. Impact of sunflower (Helianthus annuus L.) extracts upon reserve mobilization and energy metabolism in germinating mustard (Sinapis alba L.) seeds. J Chem Ecol 32:2569-83. Macias, F. A., Varela, R. M., Torres, A., Galindo, J. L. G., and Molinillo, J. M. G. 2002. Allelochemicals from sunflowers: chemistry, bioactivity and applications, pp. 73-87, in: Inderjit and A.U. Mallik (eds.), Chemical Ecology of Plants: Allelopathy in Aquatic and Terrestrial Ecosystems. Birkhauser Verlag, Basel, Boston, Berlin. Oracz K, Bailly C, Gniazdowska A, Come D, Corbineau F, Bogatek R. 2007. Induction of oxidative stress by sunflower phytotoxins in germinating mustard seeds. J Chem Ecol 33:251-64.

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Responses of field grown maize plants to phosphate solubilizing microorganisms under varying intensities of water deficit stress *S. M. R. Ehteshami, **M. Aghaalikhani, ***K. Khavazi *Member of Young Reasearchers Club of Islamic Azad University and Ph.D Student of Tarbiat Modarres University, Tehran, Iran **Tarbiat Modarres University, Tehran, Iran, ***Soil and Water Research Institute, Tehran, Iran The effect of seed inoculation by phosphate solubilizing microorganisms on growth, yield and nutrient uptake of maize (Zea mays L. SC. 704) was determined in a field experiment. Positive effect on plant growth, nutrient uptake, grain yield and yield components in maize plants was recorded in the treatment receiving mixed inoculum of Glomus intraradices (AM) and Pseudomonas fluorescens (Pf). Addition of dual-inoculation treatments, seed inoculation with AMF positively affected the measured parameters. The dual inoculation treatment significantly increased grain yield, yield components, harvest index, grain N and P, soil available P, root colonization percentage and WUE under water deficit stress. In some of investigated charadteristics under well-watered conditions, chemical fertilizer treatment was higher than double inoculated treatments, but this difference was n’t significant. Our results showed in contrast to the inoculated treatments with AM+Pf and AM, the application of alone Pf caused a comparatively poor response. Therefore, this microorganism needs to a complement for its activity in soil. All of measured parameters in inoculated treatments were higher than uninoculated treatments under water deficit stress conditions. Furthermore, the investigated characteristics of co-inoculated plants under severe water deficit stress conditions were significantly lower than co-inoculated plants under well-watered and moderate-stressed conditions, that this results suggest that these microorganisms need to time until fix in soil themselves. The present finding showed that phosphate-solubilizing microorganisms can interact positively in promoting plant growth. As well as P uptake of maize plants, leading to plant tolerance improving under water deficit stress conditions.

Does nitric oxide take part in the recognition of biotrophic and necrotrophic pathogen by potato tissue? * J. Floryszak-Wieczorek, ** Z. Górski, *** G. Milczarek, * M. Arasimowicz * Department of Plant Physiology, The August Cieszkowski Agricultural University, Wo³yñska 35, 60-637 Poznañ, Poland ** Institute of Chemistry and Technical Electrochemistry, Faculty of Chemical Technology, Poznañ University of Technology, Piotrowo 3, 60-965 Poznañ, Poland *** Institute of Chemistry and Technical Electrochemistry, Poznañ University of Technology, Piotrowo 3, 60- 965 Poznañ, Poland Introduction: The role of NO was examined in the stimulation of signalling pathways (ethylene or salicylic acid) and the activation of host programmed cell death - PCD in potato leaves subjected to infection by biotrophic and necrotrophic pathogens. Methods: Experiments were conducted on two cultivars of potato leaves (Solanum tuberosum L.), ‘Bintje’ – susceptible and ‘Bzura’ – resistant to Phytophthora infestans (biotroph) and Botrytis cinerea (necrotroph). NO detection and quantification by the electrochemical method, ultraweak photon, ethylene and salicylic acid and PCD using the TUNEL assay were analysed after inoculation. Results: NO generation, similarly as biophoton emission, was detected immediately after inoculation, but the character of this overproduction was different for both cultivars and pathogens. In the resistant ‘Bzura’ – Phytophthora infestans interaction a very strong 2-stage NO generation was observed, which lasted for at least 6 h and was manifested in PCD (TUNEL positive reaction). In the ‘Bzura’ – Botrytis cinerea system the course of NO production was similar, but it was 4 times lower and cells did not exhibit PCD. In susceptible ‘Bintje’, both the biotroph and necrotroph inoculation caused a one-stage, very weak NO burst and the TUNEL negative reaction. In the defense response of potato against the necrotroph and the the biotroph no selective stimulation of signalling pathways via ethylene or salicylic acid observed, as the highest level of both endogenous signals after inoculation was recorded in the ‘Bintje’- Phytophthora infestans system.

Conclusions: Potato tissues are probably capable of distinguishing signals coming from biotrophic or necrotrophic pathogens, which is manifested in the varied rate and intensity of early NO generation, which leads to the stimulation of PCD and signalling pathways tuning to resistance.

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Impact of the drought stress on changes of spectrofluorescence parameters in maize hybrids differing in drought tolerance M.T. Grzesiak, S. Grzesiak, A. Skoczowski Institute of Plant Physiology. PAS, Niezapominajek str. 21, PL 30-239 Cracow, Poland Introduction: The drought stress decreases crop yield by affecting the structural and functional status of the photosynthetic apparatus. Increase in chlorophyll fluorescence during drought stress is connected with starting of protective mechanisms in plant leaves. In this study spectrofluorescence method was used to estimate differences between drought resistant and drought sensitive maize hybrids in blue, green, red and far-red fluorescence. Methods: The experiment was carried out in phytotronic air-conditioned cabinet at 23/18 °C (±1 °C) day/night temperature, day length 14 h and relative humidity (RH) 65 %.Two single cross maize hybrids (drought sensitive Ancora and drought resistant Tina) were grown in pots filed mixture of garden soil, peat and sand (1:1:3 v/v). Three weeks after sowing the drought treatment (35 % field water capacity) was applied during 14 days. The luminescence spectrofluorymetr Parkin-Elmer LS50B was used to register fluorescence emission and excitation spectra. Results: Soil drying caused increase in blue, green, red and far-red fluorescence. Both in resistant and sensitive strains the increase in fluorescence in particular spectrum ranges was higher in case of older leaves then in case of younger leaves. Compared to control seedlings subjected to 7 or 14-day drought showed higher increase in case of drought sensitive hybrid Ancora then in drought-resistant Tina. After 7 days of recovery in case of hybrid Tina faster removal of harmful effect of drought was observed because fluorescence parameter measured for this hybrid were most similar to control values. Conclusions: Spectrofluorescence measurements of the chlorophyll fluorescence are a sensitive and rapid method determination of effect of drought stress on injury of photosynthetic aparaturs. Drought is responsible for damage of PSI and PSII and reduction their activity is connected with increase emission of fluorescence. The increase of emission of blue and green fluorescence was connected with accumulation of phenolic compounds in epidermal cells, which function as UV-filter for the mesophyll tissues. The increase of red fluorescence was connected with loss of chlorophyll content in stressed leaves and light conversion. Acknowledgements: This study was supported by grant from KBN no 2 P06A 001 26

Direct and after effects of drought on leaf water potential and gas exchange in triticale and maize M.T. Grzesiak*, S. Grzesiak*, A. Skoczowski*, A. Rzepka** Institute of Plant Physiology. PAS, Niezapominajek str. 21, PL 30-239 Cracow, Poland* Dept. of Plant Physiology, Pedagogical Academy, 2 Podbrzezie str., PL 30 054, Cracow, Poland** Introduction: Changes of leaf water content and gas exchange are very sensitive physiological processes to a drought stress. Reductions in leaf water potential result in photosynthetic competence in many plant species. Under mild drought, decreases in photosynthesis are generally considered to be the result of reduced availability of carbon dioxide due to stomatal closure. However when drought prolongs decrease of photosynthesis is controlled by “non-stomatal” mechanisms of gas exchange connected with damages of mesophyll cells, membranes and chloroplasts, decrease in chlorophyll content and disturbances in processes of assimilates synthesis and transport.

Methods: Experimental plants were grown in air-conditioned phytotron growth cabinets. Germinated grain was placed on the polystyrene foam plates in hydroponic containers. Conditions of simulated drought (from -0.01 to -2.45 MPa) in hydroponic cultures were obtained by using mannitol. After 21 days of seedlings growth in control conditions (yS= -0.013 MPa) the 14-day-long drought were established. After 7 or 14 day of drought control conditions were re-established for 7 day. Results: At -0.013 MPa significant differences in leaf water potential (y), net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs) and internal CO2 concentration (Ci) of drought resistant and sensitive triticale and maize genotypes were not found. Together with the increase of the mannitol solution concentration the impact of drought on transpiration rate (E) and stomatal conductance (gs) for drought sensitive genotypes became lower than for drought resistant ones was observed. Inversely, impact of drought on water potential (y) was

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higher than for resistant ones. From 1 to 3 days of drought for resistant genotypes higher decrease in photosynthesis net rate (Pn) than in sensitive ones was observed. Under prolonged drought (from 5 to 14 days) and simultaneously more severe level of drought decrease in photosynthesis rate was lower comparing to sensitive ones. Changes in water potential (y), photosynthesis rate (Pn), transpiration rate (E) and stomatal conductance (gs) caused by drought in case of genotypes differing in the drought susceptibility were similar for triticale and maize. Differences in drought influence between triticale and maize genotypes were observed in measurements of internal CO2 concentration (Ci)). Comparing to control plants, increase of Ci for triticale genotypes and a decrease of that for maize genotypes was observed.

Conclusion: Obtained results of measurements of water potential and leaf gas exchange parameters indicate, that one of the physiological reasons of different susceptibility to drought between sensitive and resistant genotypes is more efficient protection of tissue water status by resistant genotypes by higher decrease in stomatal conductance and limiting of transpiration rate compared to sensitive ones. Other reason might be, observed in case of resistant genotypes during the recovery, more efficient removal of detrimental effects of drought.

Response to drought stress of triticale and maize genotypes differing in drought tolerance M.T. Grzesiak*, T. Hura*, A. Rzepka**, K. Hura***, A. Skoczowski*, S. Grzesiak* Institute of Plant Physiology. PAS, 21 Niezapominajek str., PL 30-239 Cracow, Poland* Dept. of Plant Physiology, Pedagogical Academy, 2 Podbrzezie str., PL 30 054, Cracow, Poland** Dept. of Plant Physiology, Agricultural University, 21 Av A. Mickiewicza, PL 31 120 Cracow *** Introduction: Visible syndrome of plant exposure to drought in the vegetative phase is a leaf wilting, decrease of plant height, number and area of leaves and delay in occurrence of buds and flowers. Invisible effects are injuries of cytoplasmatic membranes, disturbances in a water status of different organs and decrease in the chlorophyll content. Changes in a tissue water status occur after few hours from beginning of drought, however, loss of membranes permeability and chlorophyll content occur later, but they are often irreversible, especially in conditions of severe and prolonged exposure to drought. These changes depend on a species, level and duration of drought, growth phase and plant age. Methods: The experiment was carried out on 2 spring triticale (x Triticosecale Wittmack) breeding strains and 2 maize (Zea mays L.) single cross hybrids. Chosen genotypes were differing in the drought susceptibility index (DSI) Experimental plants were grown in air-conditioned growth cabinets. Plants were grown in the plastic pots filled with mixture of soil, peat and sand and till 28th day after sowing plants maintained well-watered (65 % of soil field water capacity – FWC). Subsequently, drought treatment (30 % FWC) was started and applied for 7 or 14 d. After this period, for the next 7 days well watering conditions were re-established. Results: Direct and after effects of soil drought for 7 d and 14 d were examined on a seedlings dry matter, leaf water potential (y), leaf injuries index (LI) and chlorophyll (Chl) content of drought resistant and sensitive triticale and maize genotypes. Drought caused higher decrease in number of developed leaves and dry matter of shoots and roots in sensitive genotypes than in resistant ones. Soil drought caused lower decrease of y in the triticale than in maize leaves. Influence of drought on the Chl b content was considerably lower than on the Chl a content. In triticale the most harmful drought impact was observed for physiologically younger leaves and in case of maize the older ones. A period of 7-day-long recovery was to short for a complete removal an adverse influence of drought. Conclusions: Our results indicate that differences between drought resistant and sensitive triticale and maize genotypes have occurred in response to direct and post drought influence estimated by its impact on the above-ground and the root dry matter, leaf water potential, leaf injury, and leaf Chl content. These differences were small, but when drought remained for a longer period, it may suggest that it is one of the reasons of genotypical variability of drought tolerance. Acknowledgements: This study was supported by the State Committee of Scientific Investigation (KBN) grant no. 2P06A 001 26.

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Effect of drought stress on changes in chlorophyll fluorescence and spectrofluorescence emission and excitation of leaves of triticale and maize genotypes differing in drought tolerance M.T. Grzesiak*, T. Hura*, A. Rzepka**, W. Filek***, K. Hura*** and A. Skoczowski * *The Institute of Plant Physiology. PAS, 21 Niezapominajek str., PL 30-239 Cracow, Poland **Dept. of Plant Physiology, Pedagogical Academy, 2 Podbrzezie str., PL 30 054, Cracow, Poland *** Dept. of Plant Physiology, Agri. University, 21 Av A. Mickiewicza, PL 31 120 Cracow, Poland. Introduction: In 1931 Kautsky and Hirsch discovered, that plant tissue subjected to UV-A irradiation or blue light emitted red fluorescence. Kinetics of changes in fluorescence emission of leaves adapted to darkness in preliminary phase reflects photosynthesis induction and its parameters are inversely correlated to photosynthesis. In recent years, techniques based on the fluorescence phenomenon have become ubiquitous in plant physiology studies. To obtain a full picture of the response of plants to their environment fluorescence methods are combined with other techniques, in particular, with gas exchange measurements. Among fluorescence techniques the spectrofluorimetric methods are used to study the blue, green and red fluorescence emitted by plant leaves. Methods: Experimental plants were grown in air-conditioned growth cabinets. Plants were grown in Mitscherlich pots filled with mixture of soil, peat and sand. Drought treatment (30 % FWC) was started and applied for 7 or 14 days. After this period, for the next 7 days in case of seedlings earlier subjected to drought, well watering conditions were re-established. Measurements: The photochemical efficiency of PS II photochemistry was determinate with application of PSM. Fluorescence emission spectra of red fluorescence were recorded using a Perkin-Elmer LS 50B spectrofluorimetric. The fluorescence yield was measured for the 4 fluorescence bands of plants at 450, 520, 690 and 740 nm with the excitation wavelength from 250 to 400 nm. Results: An influence of the drought on changes of parameters of chlorophyll a fluorescence and a leaf fluorescence excitation spectrum in the main fluorescence bands (F450, F520, F690, F740) in drought resistant and sensitive genotypes of triticale and maize was compared. In control treatments (C) between examined genotypes some similarities and differences in chlorophyll a fluorescence and spectrofluorescence emission and excitation of leaves occurred. For triticale Fo, Fm and Fv means were higher than for maize, however Fv/Fm was higher for maize than for triticale. Also in control plants differences between maize and triticale were observed in fluorescence measurements at wavelengths of 440 and 520 nm, however for wavelengths 690 and 740 nm differences were insignificant. Drought (D) caused significant decrease in Fv and Fv/Fm. After recovery (D/R) chlorophyll fluorescence parameters mainly did not attain values observed for control plants except Fv/Fm in both triticale genotypes. Drought (D) caused the increase in emission and excitation of leaves in F450, F520 and F690 fluorescence range and this increase was more distinct for drought sensitive genotypes. After recovery (D/R) chlorophyll fluorescence in blue, green, red and far-red range mostly did attain obtained values of control plants. Drought (D) caused significant increase in F690/F740 ratio comparing to control, however in F450/F690 ratio F450/F520 ratio obtained differences were smaller and mostly statistically insignificant. For triticale, highest increase in F690/F740 ratio was observed in case of 4th and 7th leaf for resistant genotype and contrarily in maize for the sensitive one. Conclusions: This research demonstrates that fluorescence emission spectra of leaves can be successfully applied for detection of water stress in plants. Acknowledgements: This study was supported by the State Committee of Scientific Investigation (KBN) grant no. 2P06A 001 26.

Salt tolerance induced in barley by Piriformospora indica B.D. Harrach*, J. Fodor*, B. Barna*, A. Skoczowski** * Plant Protection Institute, HAS, P.O. Box 102, H-1525, Budapest, Hungary ** The Franciszek Górski Institute of Plant Physiology, PAS, Niezapominajek 21, 30-239 Cracow, Poland

Piriformospora indica is an endophytic fungus that promotes growth of colonised plants. Salt-sensitive barley (Hordeum vulgare L.) plants were inoculated with P. indica and, as a result, became salt tolerant as demonstrated by calorimetric and gas chromatographic studies. Salt treatment caused increases in lipid peroxidation and the degree of unsaturation of the fatty acids (double bond index, DBI). Furthermore, rate of metabolic heat production decreased under saline conditions. However, the high NaCl-induced responses were not identified in P. indica-inoculated plants. Moreover, the growth promoting activity of P. indica was detected in barley even in the presence of 300 mM NaCl. Our results unequivocally demonstrate that P. indica can induce salt tolerance in barley. Since

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this fungus can be grown axenically, it has a high potential for protecting plants against salt stress in arid and semiarid areas.

Differences in response to drought stress between triticale and maize plants expressed by the leaf gas exchange and spectrofluorimetric methods T. Hura*, M.T. Grzesiak*, K.Hura**, S. Grzesiak*, A. Rzepka*** *Institute of Plant Physiology, PAS, Niezapominajek 21, 30-239 Kraków, Poland **Dep. of Plant Physiology, Agricultural University, Pod³u¿na 3, 30-239 Kraków, Poland ***Dep. of Plant Physiology, Pedagogical Academy, 2 Podbrzezie, 30-054 Kraków, Poland Introduction: In recent years, techniques based on the fluorescence phenomenon have become ubiquitous in plant physiology studies. To obtain a full view of the response of plants to their environment fluorescence methods are combined with other techniques, in particular, with gas exchange measurements. Among fluorescence techniques the spectrofluorimetric methods are used to study the blue, green and red fluorescence emitted by plant leaves. Methods: Plants of spring triticale (x Triticosecale Wittmack) and maize (Zea mays L.) were grown in an air-conditioned greenhouse chamber. From the 12th day after sowing plants were maintained well-watered - 65 % FWC to allow them to adjust to the environment before drought treatment was imposed. Subsequently, drought treatment (30 % FWC) was started and applied for 14 days. After this period, for the next 14 days in case of seedlings earlier subjected to drought, well-watering conditions were re-established. Results: The studies were carried out in order to estimate differences in the physiological state between triticale and maize plants subjected to drought stress followed by rehydration. The physiological state of the plants was evaluated by measurements of the leaf water potential, net photosynthesis, transpiration and stomatal conductance. Spectrofluorimetric methods to study the blue, green and red fluorescence were applied as well. We have observed that the soil drought induced a harder water loss in triticale leaves than in maize and consequently caused larger injuries to the photosynthetic apparatus. Moreover, triticale plants recovered slower than maize plants during the rehydration phase. The effect was probably connected with the higher functional and structural disorganization of the photosynthetic apparatus observed during drought stress in triticale. Water stress is responsible for damages to photosystem PS II. The worse light utilization in the photosynthetic light conversion was recorded as an increase in the intensity of the red fluorescence. Drought stress induced a strong increase in the intensity of the blue and green fluorescence in the studied species and it was still higher in maize plants during the first day of rehydration. Increase in the intensity of the blue and green fluorescence in maize is the effect of the photoprotection mechanism which prevent damage to PS II through utilization of the excess energy. Conclusions: The protective mechanism enables maize to come out of light stress already during the drought stress through the removal of photoinhibitory injuries. Triticale cannot synthesis organic compounds efficiently, as maize does, because exhibited the lower carboxilation activity during drought periods. This less effective photoprotective mechanism in triticale causes greater photoinhibitory injuries to occur and consequently it takes longer for triticale to remove injuries to the photosynthetic apparatus in comparison with maize.

The application of spectrofluorimetric methods for studying the different responses soil drought in triticale and maize plants T. Hura *, K. Hura **, M.T. Grzesiak* *Institute of Plant Physiology, PAS, Niezapominajek 21, 30-239 Kraków, Poland **Dep. of Plant Physiology, Agricultural University, Pod³u¿na 3, 30-239 Kraków, Poland Introduction: In our experiment we used spectrofluorimetric methods to study physiological state of triticale and maize plants during and after drought stress. Measurements of the blue, green and red fluorescence can be used to detect, follow and define plants responses to stress conditions. The blue and green fluorescence originates from plant phenolics, primarily from ferulic acid covalently bound to carbohydrates of epidermal cell walls. Phenolic compounds may function as UV-filter for the leaf mesophyll and have possibilities to change properties of light falling on leaf throughout absorption UV radiation and its transformation to the blue and/or green fluorescence. The origin of the red fluorescence is the protein-bound chlorophyll a of the mesophyll cells.

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Methods: Plants of spring triticale and maize were grown in air-conditioned greenhouse chamber. In the experiment from 12 day after sowing plants maintained well-watered (65 % of FWC) and drought treatment (30 % FWC) was started and applied for 14 days. After this period, for the next 14 days in case of seedlings earlier subjected to drought, well watering conditions were reestablished. Fluorescence emission spectra of red fluorescence were measured using a Perkin-Elmer LS 50B spectrofluorometer. Results: We have found that the soil drought induced higher water loss in triticale leaves than in maize and consequently caused larger injuries of the photosynthetic apparatus. We noted that triticale plants recovered slower than maize plants during rehydration phase, what we connect with higher functional and structural disorganization of the photosynthetic apparatus. Drought stress induced strong increase in emission of the blue and green fluorescence in studied species. The values of intensity of the blue and green fluorescence during the first day of rehydration were still higher in maize plants. The increase of emission of blue and green fluorescence is connected with accumulation of the phenolic compounds in plant leaves which can function as UV-filter for the leaf tissues. Differences in the ability to recovery are connected with presence of phenolics in leaf tissues which can act as photoprotectors and can be involve in defense mechanism under drought stress conditions. It would be of value to determine whether the drought resistance of the crop plants might be connected with phenolic content especially with concentration of ferulic acid as main origin of the blue-green fluorescence. Conclusion: We suggest that adaptation to drought may depend on different factors, including the ability to sustain high levels of phenolics in leaf tissues.

Physiological improvement of salt tolerance in wheat (Triticum aestivum L.) through seed priming M.T. Javed, S.M.A. Basra, I. Afzal Department of Crop Physiology, University of Agriculture, Faisalabad-38040 Pakistan Introduction: Soil salinity is considerable problem with adverse affect on physiological and metabolic processes, finally diminishing growth and yield (Ashraf and Harris 2004). It was hypothesized that seed enhancement (seed priming) could increase salt tolerance in wheat (Afzal et al. 2005). Materials and methods: A pot study was conducted in net house to determine the influence of pre-sowing seed treatment with distilled water, inorganic salts (50 mM CaCl2, CaSO4, NaCl) and 50 ppm ascorbate on emergence and yield of spring wheat cultivars MH-97 (intolerant) and SARC-1 (tolerant) under saline (15 dSm-1) and non-saline (2.58 dSm-1) conditions. Results: Ascorbate priming for 12h maximally increased final emergence percentage, germination index, fresh and dry weight in both cultivars as compared to all pre-sowing seed treatments including control. Furthermore priming with ascorbic acid for 12h also has promoting effect on grain yield by increasing spike length, number of spikelets per spike and 100 rains weight in both cultivars. Concentrations of Na+ and K+ in seedlings changed significantly obtained after priming. However, Na+ was highest in seedlings raised from seeds primed with NaCl whereas the concentration of K+ was highest in the seedlings primed with CaSO4. In addition, seedlings obtained from the seeds primed with CaCl2 had highest level of Cl- in seedlings followed by those primed with NaCl and CaSO4. All pre-sowing seed treatments significantly affected activities of antioxidant enzymes (superoxide dismutase and catalase) in both cultivars during stress conditions. The salt-tolerant cultivar SARC-1 had a better protection against reactive oxygen species (ROS) as shown by increased SOD and CAT activities under salt stress. Conclusion. Finally it can be concluded that all the per-sowing seed treatments were effective in alleviating the effect of salt stress in both cultivars, however priming with 50 ppm ascorbate maximally improved salt tolerance in wheat References Afzal I., Basra S.M.A., Iqbal A. 2005. The effects of seed soaking with plant growth regulators on seedling vigor of wheat under salinity stress. J. Stress Physiol. Biochem. 1: 6-14. Ashraf M., Harris P.J.C. 2004. Potential biochemical indicators of salinity tolerance in plants. Plant Sci. 166: 3–6. Acknowledgement: This work was supported by Higher Education Commission (HEC) of Pakistan under “Induction of salt tolerance in wheat (Triticum aestivum L.) with seed invigoration techniques” project.

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Cortical microtubule disorientation is connected with root cell growth in clinorotated Brassica rapa seedlings I. M. Kalinina Cell Biology Department, Institute of Botany NASU, 2, Tereschenkivska Street, 01004 Kiev, Ukraine Introduction: The results of experiments performed under conditions of microgravity or under its simulation on the horizontal clinostat with seedlings of various species have revealed a definite role of gravity as an ecological factor in the processes of plant morphogenesis, growth, and development (Kordyum 1997). Methods: To determine whether the tubulin cytoskeleton is participating in gravisensitivity of cells, which are not specialized to perceive gravity stimulus, we made immunofluorescence labeling of microtubules with anti-a-tubulin antibodies (Sigma, USA) and measured cell length in the different growth zones in roots of 5-day old Brassica rapa in control and under clinorotation. Results: Confocal microscopy (LSM 5 Pascal, Zeiss) investigation showed no differences in the spatial organization of endoplasmic microtubules and in nucleus topography in control and under clinorotation. Cortical microtubules in root epidermal and cortical cells undergo the series of changes of their pattern according to the developmental stages of cells during their differentiation. In the distal elongation zone of control roots cortical microtubule arrays were organized transversely to the axis of root growth. The alterations in cortical microtubule orientation under clinorotation were revealed only in the distal elongation zone. Under clinorotation, cortical microtubules looked more randomly organized then in control. Simultaneously with preservation of transversal pattern a number of disordered single microtubules were observed. Cell shape depends on the ordering of dynamic cortical microtubules. In the root cortical microtubules ordering the transition from the isodiametric cellular growth distinctive for the distal elongation zone to the anisotropic cellular growth in elongation zone via providing spatial information for cellulose microfibril synthesis (Baskin 2001). Measurement of root cells in the different growth zones demonstrated the significant decrease in cell length only in the central elongation zone of clinorotated seedlings. Conclusions: It is suggested that the declining in anisotropic growth is connected with microtubule disorientation under clinorotation. References Kordyum E.L. Biology of plant cell microgravity and under clinostating // Int. Rev. of Cytology. -1997. - 171. P. 1-72. Baskin T.I. On the alignment of cellulose microfibrils by cortical microtubules: A review and a model // Protoplasma. -2001. -215. - P. 150-171.

Stability and plasticity of plant ontogenesis E.L. Kordyum Institute of Botany, National Academy of Sciences of Ukraine, Kyiv, Ukraine, [email protected] The question concerning in what manner an organism maintains its stability under the influence of the unfavorable environment is briefly reviewed. The system’s stability is postulated to be provided by the ability of its components to lability in certain limits, and so phenotypic plasticity is considered as a tactic way for realization of ontogenesis strategy. From the point of view that phenotypic plasticity is the manifestation of operative adaptation, i.e. adaptation in ontogenesis, phenotypic plasticity is supposed to be performed within the limits of reaction physiological norm on the basis of metabolic and hormonal regulation of gene expression. In this paper, we consider stability and plasticity of some traits of structural and functional organizations, particular at the cellular level, of Alisma plantago-aquatica L. and Sium latifolium L. two ecological forms growing in the natural conditions of different water accessibility. A signal role of water regime in formation and structure of the root system in aerial-aquatic plants is demonstrated. Since the terrestrial plants of these aerial-aquatic species perform a “reproductive imperative”, i.e. flower and bear fruits giving viable seeds, a series of revealed changes in the plant structural and functional organization may be attributed to the adaptive mechanisms at the cellular and organism levels to moderate water deficit. It is discussed the current concepts of two strategies of adaptation process: 1) fast acclimation in a response to daily or seasonal fluctuations of ecological factors as well as to short-term action of a surplus dose of any factor or its minimization (stress) and 2) long-term adaptation to the chronic moderate influence

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of unfavorable changes of ecological factors. A possible role of epigenetic heredity, different forms of which are widely spread among plants, in adaptation of wild plants to separate ecological niches, the periphery of their natural areas or being introduced is discussed. The propounded ideas open new approaches for research of genome differential activity and its phenotypic manifestation in the stress-reactions and operative adaptation of plants to the chronic influence of environmental unfavorable changes.

Induced phenylpropanoid metabolism under irradiation/allelochemical stresses M. Koz³owska, M. Pietrowska-Borek, A. JóŸwiak Department of Plant Physiology, August Cieszkowski Agricultural University of Poznañ, Poland Introduction: Under natural conditions plants experience many disadvantageous environmental factors simultaneously (Gong et al. 2001, Glombitza et al. 2004). The stresses may bring about various responses, which could be additive or antagonistic. Elucidation of the interaction of evaluated UV-B radiation and allelochemical effects would help us to understand the potential impact of plant cross tolerance. This report refers to the mechanism of co-operation or interaction between both stresses in terms of phenylpropanoid metabolism. Methods: Arabidopsis thaliana and a crop plant (Cucumis sativus ‘Dar’) were grown in a growth chamber under phenolic acid treatment conditions (p-coumaric and ferulic at 0.1 mM and 1 mM) and exposed to UV-B (16 kJ m2 d-1). The activities of 4-coumarate-CoA ligase [4CL] (Knobloch and Hahlbrock 1977) and the level of anthocyanins (Wang et al. 2000) and colourless or yellow flavonoids (Schnitzler et al. 1997) were determined. Results: Allelochemicals under lower doses slightly induced phenylpropanoid metabolism, but the higher level of phenolic acids decreased the activity of 4CL and the accumulation of flavonoids. The effect depended on the type of the allelochemical substance and on its application method; in the organic medium, hydroponic or vermiculite. High induction of phenylopropanoids was found under UV-B stress and under the simultaneous effect of both factors. In cucumber only the colourless or yellow flavonoids were recorded and induced. Conclusions: The effect of combined stresses and the elucidation of the interaction brings about an ambiguous response. Allelochemical stress seems to prevent the damage caused by elevated UV-radiation, since both are involved in the induction of phenylpropanoid accumulation, although with varying intensity. References Gong M., Chen B., Li ZG., Guo LH. 2001. Heat-shock-induced cross adaptation to heat, chilling, drought and salt stress in maize seedlings and involvement of H2O2. J. Plant Physiol. 158: 1125-1130. Glombitza S. et al. 2004. Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism. Plant Mol. Biol. 54: 817-835. Knobloch K.H., Hahlbrock K. 1977. 4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm. Partial purification, substrate specificity and further properties. Arch. Biochem. Biophys. 184: 237-248. Wang H., Arakawa O., Motomura Y. 2000. Influence of maturity and bagging the relationship between anthocyanin accumulation and phenylalanine ammonia-lyase (PAL) activity in Jonatan apples. Post. Biol. Tech. 19: 123-128. Schnitzler J.P., Jungblut T.P., Feicht C., Kofferlein M., Langebartels C., Heller W., Sandermann H. 1997. UV-B induction of flavonoid biosynthesis in Scot pine (Pinus sylvestris L.) seedlings. Trees 11: 162-168.

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Antioxidant system activity increase by synthetic compounds in corn seedlings under the salt stress conditions I.M. Kurylenko, T.O. Palladina Institute of Botany of NASU, 2, Tereschenkivska Str., Kyiv, 01601, Ukraine Introduction: Oxidative stress appearance in plant cells is universal response of living organisms on negative factors including soil salinity. Antioxidant defense system keeps the prooxidant-antioxidant status of plant tissues in balance. The aim of present investigation was to elucidate the role of plant antioxidant enzymes in adaptation to salt stress conditions and effect of synthetic preparations Methyure and Ivine on their functioning. Methods: Corn seedlings (hybrid Kolektyvnyi 225 MV) were grown on Hoagland medium during 7 days and then exposed to fresh nutrient solution contained 0.1 M NaCl during 1 and 10 days. Synthetic compounds were used by seed soaking in their 10-7M water solutions. Antioxidant system functioning was estimated by superoxide dismutase (SOD) [1], catalase [2] and non-specific peroxidase [3] activity. Results: Salt exposition during 1 and 10 days increased SOD activity in root and leaf tissues of corn seedling. 1-day salt stress induced decrease of catalase and non-specific peroxidase activity in corn tissues whereas 10-day one increased it. Seed treatment by synthetic compounds increased antioxidant enzyme activities in roots and leaves of 0.1 M NaCl-exposed seedlings, at that Methyure effect was more pronounced and prolonged than Ivine one. Conclusions: SOD plays the key role in antioxidant system of corn seedlings exposed to 0.1 M NaCl whereas catalase and non-specific peroxidase reveal their defense property at long-term salt stress only. Stimulation of antioxidant enzymes by Methyure and Ivine facilitates the adaptation process to salt stress conditions. Revealed protective role of synthetic compounds can be used for plant salt-tolerance increase. References Giannopolitis C.N., Ries S.K. // Plant Physiol. 1977. V.59. P.309-314. Aebi H. // Meth. Enzymol. 1980. V.105. P.121-126. Nakano Y., Asada K. // Plant Cell Physiol. 1981. V.22. P.867-880.

Changes in content of photosynthetic pigments and proteins in mustard (Sinapis alba, L.) cotyledons after cycloheximide and kinetin treatment A. Machlicová, ¼. Slováková, J. Hudák Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, 842 15 Bratislava, Slovakia Introduction: Generally, antibiotics inhibit chloroplast formation from proplastids, thylacoid differentiation, induce premature senescence and chloroplast degradation (Bashan et al. 2003). Cycloheximide is an glutarimide antibiotic inhibiting cytoplasmatic proteosynthesis. Cytokinins as growth regulators stimulate cell divission, chloroplast replication, chlorophyll biosynthesis, proteosynthesis and delay plant senescence (Srinivasan et al. 2006). Materials and methods: Mustard seedling growed in solution with different cycloheximide concentration (10-5M -10-9M) and in cycloheximide with kinetin (10-4M, 10-5M). The 7-day-old mustard cotyledons were analyzed for chlorophyll content (Lichtenthaler 1987). The quantitative and the qualitative analyzes of proteins was determined (Laemmli 1970, Bradford 1976, Maniatis et al. 1989). For electron microscopy cotyledons were fixed in Karnovsky fixative, postfixed in osmium tetroxide and embeded in Durcupan ACM. Ultrathin sections were stained with uranyl acetate and lead citrate. Results: The total chlorophyll after cycloheximide treatment decreased significantly compare to control. The highest protein amount was found in cotyledons treated with 10-5M cycloheximide. When cycloheximide and kinetin were applied photosynthetic pigments inreased almost 4-fold and proteins 2-fold compare to cycloheximide sample. Chloroplasts of cycloheximide treated cotyledons have less regular shape, form small

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spurs and phytoferritin occures. After kinetin treatment plastid membrane system is better formed in comparison with plastids in cycloheximide treated cotyledons. Conclusions: Based on our experiments we could conclude that kinetin has the positive influence on photosynthetic apparatus of cycloheximide treated mustard seedlings. References Bashan, A. et al., 2003. Mol. Cell 11: 91-102. Bradford, M.M. 1976. Analyt. Biochem. 72: 248-254. Laemmli U.K, 1970. Nature 227: 680-685. Lichtenthaler, H. K. 1987. Methods Enzymol. 148:350-382. Maniatis T. et al., 1989. Cold Spring Harbor Laboratory, New York, 1847-1859. Srinivasan M. et al. 2006. J. Biosci. 31: 599–605. Acknowledgements: This work was supported by Grant agency VEGA No. 1/3288/06 and grant UK/155/2006.

Resistance mechanisms of winter cereals and forage grasses to low temperature parasitic fungi K. Marzec Department of Plant Physiology, University of Agriculture of Cracow, ul. Pod³uzna 3, 30-239 Kraków, Poland Winter survival of cereals and grasses depends mainly on the plant resistance to low temperature and to snow mould fungi. It means, that to persist winter plants have to be tolerant to different kind of stress: biotic (mainly low temperature pathogenic fungi) and abiotic such as low temperature, snow and ice cover, freeze-induced plant desiccation or frequent freezing and thawing (Proñczuk and Zagdañska 1992). The most occurring low temperature fungus causing damages of cereals and grasses in Poland is the pink snow mould (Microdochium nivale) (Wiœniewski et al. 1997). During cold acclimation cereals and grasses become more resistant to both stresses: cold and snow mould. Older plants and these with greater number of crowns are more resistant to snow mould. Infection by Microdochium nivale induce very complex plant responce, for example: synthesis of PR-proteins (Pathogenesis-Related proteins): chitinase, b-1,3-glucanase, production of active oxygen species, synthesis of phenolics, phyotalexins, accumulation of calosis and carbohydrates and reducing of water potential (Baker and Orlandi 1995, Hammond-Kosack and Jones 1996). References Baker C. J. Orlandi E. W. 1995. Active oxygen in plant pathogenesis. Annu. Rev. Phytopathol. 33: 299-321. Hammond-Kosack K.E., Jones J. 1996. Resistance gene-dependent plant defense response. Plant Cell 8: 1773-1791. Proñczuk M., Zagdañska B. 1992. Effect of Microdochium nivale and low temperature on winter survival of perennial ryegrass. J. Phytopathol. 138: 1-8. Wiœniewski K., Zagdañska B., Proñczuk M. 1997. Interrelationship between frost tolerance, drought and resistance to snow mould (Microdochium nivale). Acta Agronom. Hungar. 45: 311-316.

The response of winter wheat plants treatmented with hyaluronic acid to drought stress M.F. Mykhalskyi Kholodny Institute of Botany, NAS of Ukraine, 2 Tereshchenkivska St., Kyiv, 10601, Ukraine Introduction: Drought disturbances water status of plants. For a regulation of plant water exchange processes large spectrum of biological active compounds are used. The hyaluronic acid is also perspective for that. It is macromolecular proteoglycan characterized by a highly polymerized chain of glucuronic acid and N-acetylglucosamine units (Tan et al. 1990). It bounds water in 1000 times greater than its own mass that is important in controlling of tissue hydration (Block and Bettlheim 1970).

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Methods: The winter wheat cultivars Smuglyanka and Podolyanka were used in greenhouse experiments. The wheat seeds were treated by water solutions of hyaluronic acid and K2SO4 before sowing. The leaves dry mass and water deficit were measured after 10 days of drought. Results: It is established, that the leaves dry mass of winter wheat seedlings was higher at the treatment by hyaluronic acid and its blend with K2SO4 by 13.0-24.0 % (cultivar Smuglyanka) and 28.8-32.5 % (cultivar Podolyanka), than in plants grown from untreated seeds. At the same time the water deficit value in the leaves of wheat cultivar Smuglyanka was lower at the treatment by hyaluronic acid by 31.7 %, than in untreated plants. In cultivar Podolyanka plants this parameter was lower by 27.7-32.4 % at treatment seeds by hyaluronic acid and its blend with K2SO4 respectively. Conclusions: Thus, observed data showed the possibility of hyaluronic acid and its blend with nutrition elements using for decrease of drought stress influence on wheat plants. References Block A., Bettleheim F. Water vapour sorption of hyaluronic acid // Biochem. Biophys. Acta. – 1970. – 201. – P. 69. Tan S.W., Johns M.R., Greenfield P.F. Hyaluronic acid – a versatile biopolymer // Aust. J. Biotechnol. – 1990. – 4, No. 1. – P. 38-43.

The role of cell walls in adaptation of plants to moderate water deficit O.M. Nedukha Institute of Botany, Tereschenkivska str., 2, 01601, Kiev, Ukraine Introduction: Cell walls, especially hydrophilic walls with intermicellar pores of 5-7 nm in diameter, play very important role for water transport. Besides that, polysaccharides composition, particularly the present of amorphous cellulose and calcium ions in apoplast have the great influence on water transport and wall rigidity that influenced not only on growth speed of cells but also on organs growth (Nedukha 2003). Therefore, the study of role of cell wall ultrastructure, localization and amount of Ca2+ in apoplast of leaf cells of Sium latifolium air-water plant in adaptation of plant to moderate water deficit was the main aim of our investigation. Methods: Transmission and scanning electron microscopy, and laser confocal microscopy were used in the experiments. Not only the integral leaves, but also the epidermis and mesophyll of first paired leaflets of Sium latifolium L. plants of two ecotypes: air-water and terrestrial have been investigated. Results: It is established that moderate water deficit lead not only to change of integral signs of ultrastructure of cell walls of adaxial and abaxial epidermal cells, but also change the cellulose content in walls of palisade and spongy mesophyll cells in leaves of S. latifolium. The changes in epidermis are expressed by ultrastructure of external epidermal walls. Besides that, the changes in redistribution and relative content of calcium ions in cell walls of leaves under influence of water deficit were discovered. The detected changes in cell walls favor of facilitate to adaptation of plant to moderate water deficit. References Nedukha O. M. Leaf: tissue, cellular and molecular levels. // In: Cellular Mechanisms of Plants Adaptation. Ed. E.L. Kordyum, Kiev: Naukova Dumka, 2003, p. 117-218.

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Physiological role of phenolic compounds in plant response to stresses * A. P³a¿ek, ** F. Dubert * Department of Plant Physiology, Faculty of Agriculture and Economics, Agricultural University, 30-239 Kraków, Pod³u¿na 3, ** F. Górski’ Institute of Plant Physiology, Polish Academy of Sciences, 30-239 Kraków, Niezapominajek 21 Phenylpropanoids are a very important group of the second metabolites. The pathway of phenolic biosynthesis is induced in plants under treatment of various stresses. Phenolic compounds act dual: they can be toxic for plant, inhibiting their growth and development, and on the other side they protect plants from stress effect. In the lecture the most important phenolics and their influence on plant metabolism will be discussed. In plant physiology phenolic compounds have been grouped into simple phenolics, tannins, flavonoids and isoflavonoids. Simple phenolic compounds involve mainly phenolic acids (coffeic, salicylic, pherulic, chlorogenic and others), vanilin and coumarins. Chlorogenic acid demonstrates antibiotic properties, while salicylic acid (SA) is a plant growth and development regulator, playing also signal role in plant defence response to numerous stresses. SA initiates synthesis of PR (pathogenesis-related) proteins, hydrogen peroxide production and controls systemic acquired resistance (SAR). Phenolic alcohols condense to lignin, which strengths cell wall and builds natural barrier against pathogen attack. Compounds like vanilin, strong aromatic, attract insects and are used in cosmetic and food industry. Phenolics play also allelopatic role secreted by roots into soil, and inhibiting germinating and growth of other plants. Coumarins show phototoxic effect and also demonstrate growth inhibitor action. Tannins protect plants from pathogens and deter preying incests. These compounds with allelopatic properties could be used natural herbicides. Flavonoids involve anthocyanins, flavons and flavonons. They are pigments of flowers and leaves, and can protect cell structures and organic compounds from cold, UV radiation and free radicals. Isoflavonoids are characterized mainly by insecticide feature. Many isoflavonoids belong to phytoalexins, specific compounds synthesized within defence mechanism against stresses. They inhibit fungal spore germination and act osmotically to penetrating hyphae. Moreover, these compounds may imitate a steroid molecules joining to specific steroid receptors disturbing numerous metabolic processes. Among the most known phytoalexins pisatin, phaseolin and medicarpin are mentioned.

Stress signaling role of salicylic acid to cadmium-induced oxidative stress L.P. Popova*, A. Krantev *, R. Yordanova*, T. Janda**., G. Szalai** . * Acad. M. Popov” Institute of Plant Physiolpgy, Bulg.Acad. Sc., Acad. G. Bonchev str., bl. 21, 1113 Sofia, Bulgaria, ** Agricultural Research Institute, Hung. Acad. Sci., H-2462, Martonvásár, POB 19, Hungary. Introduction: Salicylic acid (SA) as a potent signaling molecule in plants is involved in eliciting specific responses to biotic and abiotic stresses. It has been shown that SA provides protection in maize and winter wheat plants against low-temperature stress, induces termotolerence in mustard seedlings or modulates plant responses to salt and osmotic stresses, ozone or UV light, drought, herbicides (Ananieva et al. 2002, 2004, Apostol et al. 2006). This study was undertaken to determine the physiological and biochemical changes in maize plants treated by SA during Cd-induced stress, to investigate whether this plant regulator is involved in the induction of defense response and to test the hypothesis that the observed protection of SA on photosynthesis against Cd stress is mediated by its effect on antioxidant defense system.

Methods: Seeds of maize were sterilized and divided into two groups. One half of the seeds was soaked in 500 mM SA solution for 6 h , the other half of the seeds was soaked in water (control), and then the both groups were allowed to germinate on moist filter paper in the dark. Three-day-old, dark grown seedlings, were placed in pots filled with 0.6 L modified Hoagland solution. CdCl2 was added at a concentration of 10, 15 and 25 mM and plants were grown for additional 12 d. CO2 fixation rate, carboxylating enzymes activity were measured radiometrivally. SOD, APX, POX and CAT activities were measured spectrophotometrically. Cd content was measured by atomic absorption spectrophotometer, and SA level by HPLC. Results: Exposure of plants to Cd caused a gradual decrease in shoots and roots dry weight accumulation, the effect being higher expressed at 25 μM Cd. Pretreatment of seeds with SA alleviates the negative effect of Cd on plant growth parameters. The rate of CO2 fixation was lower in Cd-treated plants and the inhibition was partially overcome in SA-pretreated plants. A drop in the activities of RuBPC and PEPC was observed for Cd-treated plants. Pretreatment with SA alleviates the inhibitory effect of Cd on the enzymes activity. The proline production, the rates of lipid peroxidation and electrolyte leakage increased in Cd-treated plants and the values of these parameters were much lower in SA-pretreated plants. Treatment of plants with Cd decreased the activity of APX but caused over twice increase in SOD activity. Pretreatment with SA caused increase in both APX and SOD ac-

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tivity but caused a strong reduction in CAT activity. Treatment with SA prevents the high accumulation of Cd in maize roots, SA accumulation alone was higher in control than Cd-treated plants. Conclusions: The data suggest that SA might protect cells against oxidative damage and prevent photosynthesis upon cadmium toxicity. References Ananieva EA, Alexieva VS, Popova LP. 2002. J Plant Physiol 159: 685-693 Ananieva EA, Christov KN, Popova LP. 2004. J Plant Physiol. 161: 319-328 Apostol S., Szalai G., Sujbert L., Popova L. Janda T. 2006. Z. Naturforsch. 61c:734-740 Acknowledgements: This work was supported by Bulg. Sci. Res. Foundation (B 1402) and by the Hung. Sci. Res. Foundation (OTKA T046150).

The effect of high temperature stress during storage period on seed potato vigour K. Rykaczewska Plant Breeding and Acclimatization Institute, Jadwisin Division, 05-140 Serock, Poland, e-mail: [email protected] Introduction: The results of studies on the importance of seed tuber physiological ageing in potato yielding indicate that the reaction of several cultivars on the same environment conditions during vegetation, storage and pre-sprouting (Rykaczewska 2003) is diverse. Therefore a way of seed potato treatment in the period from harvest to planting should be adapted to each cultivar. The aim of the work was the determination of high temperature stress effect during storage period on tuber seed vigour of 24 potato cultivars. Methods: The tested cultivars were: Denar, Fresco, Impala, Karatop, Krasa, Velox (very early), Augusta, Delicat, Kuklik, Lady Florina, Rosalind, Vineta, Vitara (early), Adam, Bartek, Cekin, Fribona, Pirol, Roxana, Satina, Tajfun, Triada (middle early), Wist (middle late) and Inwestor (late). The high temperature stress during storage periode, about 18 °C, effected on seed tubers located in darkness and on the light. The control treatment were stored in darkness in 3 °C and four week before planting were pre-sprouted on light and temperature 18 °C. The field experiment was conducted in the year 2006. Results: It was distinguished 7 cultivars characterizing by very high tolerance to temperature stress under light conditions: Impala, Karatop, Augusta, Delikat, Bartek, Cekin i Pirol. The vigour of their seed tubers was the equal of the control treatment. The effect of high temperature stress under dark conditions was much stronger than under light conditions and only one cultivar (cv. Cekin) was very tolerant. The cultivars Fresco, Lady Florina, Fribona, Triada and Wist characterized by low vigour of mother tubers when they were located in high temperature in darkness. Conclusions: Results indicate that the rate of physiological ageing of potato seed tubers is not depending on the earliness of cultivars and that the high temperature stress under dark conditions is more stronger than under light conditions. References Rykaczewska K. 2003. Field method of assessment of the rate of potato seed tubers physiological ageing. Fragmenta Agronomica 3: 65-74

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Biological nitrogen fixation with non-legumes: An achievable target or a dogma? *S. P. Saikia, **V. Jain *Division of Medicinal, Aromatic & Economic Plants, North East Institute of Science & Technology (CSIR), Formerly Regional Research Laboratory, Jorhat – 785 006, Assam, India **Division of Plant Physiology, Indian Agricultural Research Institute, Pusa – 110 012, New Delhi, India Crop species other than legumes are not capable to fix atmospheric nitrogen, and consequently their yields are dependent upon high dosages of chemical fertilizers, which consume large amounts of energy and add to the cost of production. One way to conserve this energy and to check high production cost with environment protection too is by enhancement of biological nitrogen fixation through the genetic engineering of the genes responsible for nitrogen fixation. Various attempts have been made to extend the host range of Rhizobium from legumes to non-legumes through plant genetic manipulation. The transfer of nif genes along with others for functional nitrogen fixation was considered to be the most suitable strategy to achieve symbiotic N2 fixation in non-legumes. But the highly complicated system of gene regulation posed a great hindrance in achieving a functional N2-fixing transgenic system. Genetic engineering through biotechnological means has seen little or no success in achieving the induction of symbiosis between cereals and diazotrophs. Induction of nodulation has therefore been the main target of researchers over the past few years. Looking to the future, will we see Rhizobium-nodulated nonlegume crops ‘down on the farm’? At present it is impossible to say. Much more needs to be done before we can expect to see nitrogen-fixing nodules in, for example, a field-grown crop of oil-seed rape. However, the work of the different groups has opened the way and hopefully will stimulate further research. It is the author’s opinion that there are now sound reasons to anticipate that at least some non-leguminous field crops may also become independent of soil nitrogen. We intend to explain the reasons for this renewed optimism, against the background of knowledge accumulated in the past century that will be relevant to any ultimate success in exploiting these new approaches.

Distribution of calcium ions in Sium latifolium root cells affected by moderate water deficit M.A. Sobol, E.L. Kordyum Department of Cell Biology and Anatomy, Institute of Botany of NASU, Tereschenkivska St., 2, 01004, Kyiv, Ukraina Introduction: Resolving general biology problem of plant adaptation to unfavorable environmental conditions, it was concluded (Kordyum et al. 2003) that operative adaptation of aerial-aquatic plants to chronic moderate water deficit in the soil occurs by the way of formation of specialized cell adaptive mechanisms which determine plant ontogenesis in the conditions of the long-term stress factor action. Nevertheless, the problem of the intermediate links between the perception of external signal and the cell physiological responses remains still unclear. Methods: We investigated two ecotypes of Sium latifolium grown in the contrasting conditions of water regime, near the river shore in water and on the riverside, in the conditions of chronic moderate water deficit. Immediately after harvest, plant roots were incubated in the calcium-specific fluorescent dye fluo-4 (Molecular Probes, USA) for 25 min in darkness. Observations were carried out with a confocal microscope LSM 5 Pascal (Carl Zeiss, Germany) at the excitation wavelength 494 nm and emission wavelength 516 nm. We studied three different root zones, namely meristem, distal elongation zone (DEZ), and central elongation zone (CEZ). Data on the calcium ion distribution and quantification in cells were obtained using software “Pascal”. A fluorescence intensity correlates directly with cytosolic Ca2+ concentration at the corresponding emission wavelength (Ng, McAinsh 2003).

Results: We revealed that Ca2+ in the presence of specific fluorescent indicator were brightly fluorescent in green color in cells. The coefficient of an increase in the Ca2+ fluorescence intensity in cells of each following zone as compared to the preceding one (meristem-DEZ-CEZ) was 1.1 in plants of both Sium latifolium ecotypes. We clearly showed that, in Sium latifolium plants grown on the riverside, in the conditions of chronic moderate water deficit, the Ca2+ fluorescence intensity increased by 7 % in meristematic cells, and by 10 % in DEZ and CEZ cells in comparison with plants grown near the river shore in water. Conclusions: An increase in the Ca2+ fluorescence intensity in Sium latifolium root cells affected by chronic moderate water deficit could be involved in the signal transduction on the altered environmental conditions or in metabolic cell responses to moderate water deficit.

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Does wounding increase Pb-tolerance in sunflower seedlings?- Study of antioxidant response in primary and adventitious root systems. J. Strubiñska, M. Mroczek Department of Cell Biology, M. Curie-Sk³odowska University, Akademicka 19, 20-033 Lublin, Poland Introduction: A lot of evidence testifies that the response of plants to a combination of two different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the different stresses applied individually (Mittler 2006). Some evidence shows that stresses, i.e., wounding are able to increase another abiotic stress tolerance in plants (Capiati et al. 2007). In this work, antioxidant responses in plants treated by wounding and lead stresses simultaneously was investigated. Methods: Five-day old sunflower seedlings with two different types of root system: primary- formed from embryonic tissues and adventitious- originating from the hypocotyle after cutting off primary roots were subjected to lead acetate in doses: 0, 0.5, 2.5, 5 and 20 mg Pb dm-3 for a week and then studied. Shoot and root growth (measured as length and weight increase), lead accumulation as well as selected parameters representative of cellular oxidative damage (MDA) or protection (SOD and GSH) were investigated to compare plant stress response. Results: The comparison of plant stress response showed significant differences between plants with primary and adventitious root systems in almost all the studied parameters. Lower lead content and MDA level, higher SOD activity and GSH level as well as a higher root tolerance index were detected in the seedlings with adventitious roots. Otherwise, comparison of the results obtained for seedlings with different roots systems and similar metal tissue concentration suggests that the higher tolerance described above cannot be taken as a simple consequence of plant lead content. It can be assumed that adventitious root seedlings are more tolerant to lead. Conclusions: The results imply a unique acclimation response of sunflower seedlings to the combination of mechanical/lead stress and suggest that wounding has a protective influence against lead treatment. References Capiati D. A., País S.M., Téllez-Iñón M. T., 2007. Wounding increases salt tolerance in tomato plants: evidence on participation of calmodulin-like activities in cross-tolerance signaling. J. Exp. Bot. 57, 2391-2400. Mittler R., 2006. Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11, 15-19.

The threshold of photoinhibition as an indicator of stress state of plants N.M. Topchiy, E.K. Zolotareva N.G. Kholodny Institute of Botany, Tereschenkivska str. 2, Kyiv, 01601, Ukraine Introduction: In nature, all plants are adapted in a different way to various ecological factors (light, temperature, humidity, soil acidity, nutrients). The characteristics of ecological niches may be based on species plant composition that is plants serve as bio-indicators of environmental factors. Several phytoindicatory systems (ecological scales) were proposed by Ellenberg, Landoldt, Ramenskiy and other, in which a certain ball is assigned for each plant species with regard to certain ecological factors. Thus, in relation to light the plants vary from sciophyte (shade-requiring) to heliophyte (shade-requiring). The susceptibility of plants to light stress depends on the species, growth light conditions and other environmental factors. In general, shade plants or plants grown at low light are more susceptible to photoinhibition than sun plants or high-light grown plants. The aim of this work was to study the threshold of photoinhibition of plants different in shade-tolerant scale. Methods: Chlorophyll fluorescence parameters (Fv/Fm, F’v/F’m, qP, qN, NPQ and fPSII) were determined using XE-PAM fluorometer (Germany) under wide ranges of actinic light intensity (from 50 up to 1500 mmol (quantum) m-2 s-1). The threshold of photoinhibition was estimated by qP parameter value.

Results: The onset of light stress is defined as a significant decrease of photochemical quenching (qP) – empirically determined as < 0.6 (Öquist 1992). Chl fluorescence parameters were determined for a wide range of plants, from heliophytes (Helianthus laetiflorus) to sciophyte (Dryopteris carthusiana). Chl fluorescence data showed

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that the photoinhibition of photosynthetic apparatus of low-light grown plants takes place under lower light intensity compared to light-requiring plant species. Conclusions: An assessment of the threshold of photoinhibition by using parameter of photochemical quenching of chlorophyll fluorescence can be a useful criterion for characteristic of ecological niches of organisms. The results indicated differences in the threshold of photoinhibition in light- and shade-requiring plant species. It was proposed a new criterion for estimation of plant physiological state by using the threshold of photoinhibition. References Öquist G., W.S. Chow, J.M. Anderson. // Planta, 1992, V.182, P.450-460.

The impact of the sunflower and mustard extracts on the germination of mustards seeds M. Troæ The Franciszek Górski Institute of Plant Physiology, Polish Academy of Science, 30-239 Cracow, Poland Introduction: The target of the investigation was the future studies on allelopathy using isothermal calorimetry (Kupid³owska et al. 2006). Additionally, we take a trial to indicate the group of chemical compounds, which are responsible to allelopathic effects. Apply of FT – Raman spectroscopy was used to indicate the changes in the plant tissue caused by allelopathic compounds. Methods: The seeds of mustard were germinated on extracts from sunflower or mustard leaves. The extracts were separated on the SPE Columns and they chemical composition ware investigated by the UV – VIS spectrometry and UV – VIS spectrofluorymetry. The heat production during germination of the seeds was measured by isothermal calorimetry (Skoczowski, Janeczko 2004). The changes in the seeds, coused by various extracts, were measured by FT – Raman Spectroscopy. The significances in spectrums of solutions and seeds chemical composition ware analyzed by Cluster Analysis. Results: The heat production during seeds germination depends on the extracts. All the extracts have influence on the germination of the mustard seeds. Crude sunflowers and mustard extracts strongly inhibited germination. Other extracts influence on seeds germination in less extends or they haven’t influence. This depends mainly on the phenolic compounds, which are in the extracts (Reigosa et al. 1999). The FT – Raman Spectroscopy confirms that, the phenolic compounds have the biggest influence on the metabolism of seeds. Cluster Analysis occurs useful for analysis of differences in chemical composition of extracts and seeds. Conclusions: Results indicate that the extracts of the sunflower and mustard influence on the germination and heat production of the mustard seeds in different level. References Kupid³owska E., Gniazdowska A., Stêpieñ J., Corbineau F., Vinel D., Skoczowski A.,. Janeczko A., Bogatek R, 2006. Impact of sunflower (Helianthus annus L.) extracts upon reserve mobilization and energy metabolism in germinating mustard (Sinapsis alba L.) seeds, J. Chem. Ecol. 32, 2569-2583 Reigosa M.J., Souto X.C., Gonzales L., 1999. Effect of phenolic compounds on the germination of six weeds species, Plant Growth Regul., 28, 83-88 Skoczowski A., Janeczko A., 2004. Isothermal microcalorimetry as a tool in plant physiology, Analytical methods in plant stress biology 159-166 Acknowledgements: I thank Dr Ma³gorzata Barañska for help with FT – Raman Spectroscopy measurements.

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The effect of heavy metals on photosynthesis E.K. Zolotareva M.G.Kholodny Institute of botany, Tereshchenkivska str. 2, Kyiv, 01601, Ukraine, corresponding author: [email protected] Introduction: Plants grown under natural conditions have to face increasing anthropogenic pollution with heavy metals [1] Despite of the enormous studies that have devoted to the effects of heavy metals on plant physiological behavior, there is no clear understanding all mechanisms involved in the process. A significant effects of mercury, copper, nickel and cadmium ions on primary photosynthetic reactions are well described. A general response of plants to elevated levels of heavy metals is increased synthesis of various heat-shock proteins (Hsps) [2]. The chloroplast smHsps are produced when heavy metals accumulate in the chloroplast and damage photosynthesis and that smHsps help protect photosynthesis from excess heavy metals. It was suggested that Hsps function is to protect membranes from metal damage [3]. The aim of the present investigation is to compare the effects of various heavy metals on photosynthetic electron transfer and efficiency of the light energy transformation. Methods: Pea (Pisum sativum L.) seeds were grown on hydroponic nutrient media which was supplemented with different concentrations of CuSO4, CdCl2, HgCl2 and Ni(NO3)2 from 0.1 mM (control) to 5 mM. The PPFD of the light phase was 400 mmol m-2 s-1. Chlorophyll was estimated by extraction in 80 % acetone and its absorbance was measured with a spectrophotometer at 652 nm.Chloroplasts were isolated according to Walker et al., 1980. Light-induced oxygen exchange was measured by Clark-type electrode. To measure electron transfer rate in isolated chloroplasts 1 mM methyl viologen or 0.4 mM K3Fe(CN)6 as electron acceptor. Photosynthetic efficiency was estimated by determining the ratio of variable-to-maximum fluorescence (Fv/Fm) of dark-adapted leaves with a PAM-fluorometer (Walz, Germany). Results: All tested metals decreased photosynthetic oxygen evolution and affected the efficiency of light energy transformation. Nickel and mercury ions decreased photosynthetic electron transport also in vitro. It was shown that at longer exposure of plants (more 4 d) to elevated concentration of heavy metals photosynthetic efficiency and photochemical reactions partially repaired. Conclusions: The data support a hypothesis that if higher plants accumulated enough amount of heavy metals in photosynthetic cells a protective mechanism for photosynthetic apparatus is developed. References M. Mejáre, L. Bülow// Trends Biotechnol. 2001, V. 19, P. 67-73. J. L. Hall // J. Exp. Botany, 2002, V. 53, P. 1–11. S. A. Heckathorn et al.// Am.J.Bot., 2004, V. 91, P. 1312- 1318.

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