LITHUANIAN UNIVERSITY OF AGRICULTURE
RESPONSE OF PEA (Pisum sativumL.) DIFFERENT MORPHOTYPES TO THE EFFECT OF OZONE AND UV-B RADIATION
Summary of doctoral disertation Biomedical sciences (B000), Agronomy (06 B)
The dissertation was prepared at the Lithuanian University of Agriculture during 2004-2008.
Scientific supervisor: Prof. habil dr. Algirdas SLIESARAVIČIUS (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06B) Supervisor: Doc. dr. Asta RAMAŠKEVIČIENĖ (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06B)
Dissertation is defendet in Lithuanian University of Agriculture at the Council of Defence for the Agronomy sciences:
Chairperson: Prof. habil. dr. Rimantas VELIČKA (Lithuanian University of Agriculture, Biomedical Scien-ces, Agronomy 06B) Members: Prof. habil. dr. Honorata DANILČENKO (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06B) Doc. dr. Bronislovas GELVONAUSKIS (Plant Gene Bank, Biomedical Sciences, Agronomy 06B) Doc. dr. Irena PRANCKIETIENĖ (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06B) Dr. Tadeušas ŠIKŠNIANAS (Lithuanian Institute of Horticulture, Biomedical Sciences, Agronomy 06B)
Official opponents: Doc. dr. Sigutė KUSIENĖ (Lithuanian Forest Research Institute, Biomedical Sciences, Biology 01B) Prof. dr. Vytautas ŠLAPAKAUSKAS (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy 06B)
Defence of doctoral dissertationwill take place at the public meeting of the Concil of Agronomy Science on the 18th of September 2009 at 14 a.m. in the room 261, Central building of the Lithuanian University of Agriculture. Address: Lithuanian University of Agriculture, Studentu st. 11, Lt-53067 Akademija, Kaunas, Lithuania. Ohone/Fax.: (8-37) 752 211, (37) 397 500,
The summary of the doctoral dissertation was distributed on the 17th of August, 2009. The doctoral disesertation is available in the libraties of the Lithuanian University of Agriculture and the Lithuanian Institute of Agriculture. 2
LIETUVOS ŽEMĖS ŪKIO UNIVERSITETAS
SĖJAMOJO ŽIRNIO (PISUM SATIVUML.) SKIRTINGŲ MORFOTIPŲ REAKCIJA Į OZONO IR UVB SPINDULIUOTĖS POVEIKĮ
Daktaro disertacijos santrauka Biomedicinos mokslai (B000), agronomija (06 B)
Kaunas - Akademija, 2009
Disertacija rengta 2004 – 2008 metais Lietuvos žemės ūkio universitete.
Mokslinis vadovas: Prof. habil. dr. Algirdas SLIESARAVIČIUS (Lietuvos žemės ūkio universitetas, biomedicinos mokslai, agronomija 06 B) Konsultantė: Doc. dr. Asta RAMAŠKEVIČIENĖ (Lietuvos žemės ūkio universitetas, biomedicinos mokslai, agronomija 06 B)
Disertacija ginama Lietuvos žemės ūkio universiteto Agronomijos krypties taryboje:
Pirmininkas: Prof. habil. dr. Rimantas VELIČKA, (Lietuvos žemės ūkio universitetas, Biomedicinos mokslai, agronomija 06B) Nariai: Prof. habil. dr. Honorata DANILČENKO, (Lietuvos žemės ūkio universitetas, Biomedicinos mokslai , Agronomija (06B) Doc. dr. Bronislovas GLVONAUSKAS, (Augalų genų bankas, Biomedicinos mokslai, agronomija (06B) Doc. dr. Irena PRANCKIETIENĖ, (Lietuvos žemės ūkio universitetas, Biomedicinos mokslai, Agronomija(06B) Dr. Tadeuš as ŠIKŠNIANAS, (Lietuvos sodininkystės daržininkystės institutas, Biomedicinos mokslai, Agronomija (06B) Oponentai: Doc. dr. Sigutė KUUSIENĖ, (Lietuvos miškų institutas, Biomedicinos mokslai, biologija(01B) Prof. dr.Vytautas Antanas ŠLAPAKAUSKAS, (Lietuvos žemės ūkio universitetas, Biomedicinos mokslai, agronomijos (06B)
Disertacija bus ginama viešame Agronomijos mokslo krypties tarybos posėdyje 2009 m. rugsėjo men. 18 d. 14 val. Lietuvos žemės ūkio universiteto Centrinių rūmų 261 auditorijoje. Adresas: Lietuvos žemės ūkio universitetas, Studentų g. 11, 53067 Akademija, Kauno r., Lietuva. Tel./Fax.: (8-37) 752 254, (37) 397 500.
Disertacijos santrauka išsiuntinėta 2009 m. rugpjūčio 17 d. Disertaciją galima peržiūrėti Lietuvos žemės ūkio universiteto ir Lietuvos žemdirbystės instituto bibliotekose. 4
In the recent years, climate change, the decline of the stratospheric ozone layer, the increasing intensity of ultraviolet (UV) irradiance, acidic rains and the increase of the near-ground ozone concentration are approached as the most important anthropogenic processes which cause negative changes in the flora condition, productivity and biological diversity. These problems are especially actual because Lithuania, as the geographic centre of Europe, occupies very unfavourable position if long-distance air mass movements and the regional processes of environmental pollution be considered. Besides, in our geographical latitudes the vegetation of forest and agricultural ecosystems can be negatively affected by the global climate change to an especially large extent. Environmental models suggest that a half of 1350 European plant species will be susceptible and will become extinct until the year 2080 because of increasing temperature and precipitation (Spussana, Lüscher, 2006). Tropospheric or near-ground ozone (O3) is one of the main secondary air pollutants. Worldwide observations indicate that the ozone concentration is increasing year by year (Huttunenet al., 2002; UNECE, 2004). Because of unequal genetic and physiologic characteristics, the plants of different species as well as individuals within the same species are unequally resistant to the ozone influence (Huttunenet al., 2002). Ozone suppresses the development and growth of susceptible plant species (e.g., leguminous and cereal plants) even at low concentration. It has been confirmed that ozone decreases plant biomass (Winner, 1994), photosynthesis (Heath, 1994), carbon distribution (Pellet al., 1993), stomatal conductance (Reiling, Davison, 1995). Near-ground ozone acts as a strong oxidant which causes oxidative stress in plants, damages cell membranes and plasma, unbalances the flows of metabolism and information (Heath, 1994). The effect of ultraviolet radiation on living organisms is the stronger the shorter ray wavelengths are. The thickness of the ozone layer most determines the flow of UV-B rays, therefore when ozone layer is decaying this portion of the ultraviolet radiation spectrum creates the greatest danger to the vegetation (Helsper et al., 2003; Krizek, 2004). According to the research data of various authors, the effect of UV-B radiation on plants is quite many-sided. The radiation which is higher than normal causes the damages of plant cells. These are damages on DNA, membranes and the photosynthetic systems as well as a negative effect on phytohormones (Rozemaet 5
al., 1997; Jansenet al., 1998; Hollosy, 2002). The changes and damages of molecules unavoidably alter other processes (e.g., gene activity, metabolism, photosynthetic intensity) which affect plant growth (Brosche, Strid, 2003). The ability of different plant species (varieties) to adapt to changing climatic and environmental conditions becomes recently one of the most actual scientific problems which has a certain practical importance. The investigation of the integrated effect and the prognosis of possible further changes of the flora condition, productivity and biological diversity form a task which is not only of especial importance but also of particular complexity. Work hypothesis.Different pea (Pisum sativum L.) morphotypes respond differently to various anthropogenic factors. This can be related with an alteration of plant metabolism and with changes of photosynthetic pigments and biochemical indexes. Work aim and the tasks.To investigate the effects of ozone and UV-B radiation on different pea (Pisum sativumL.) morphotypes under changing climatic conditions and to measure plant reaction to the effects of ozone and UV-B radiation. 1. To investigate the effects of ozone and UV-B ra-diation on the morphometric parameters of different pea morphotypes under changing climatic conditions; 2.To investigate the effects of ozone and UV-B radiation on the photosynt-hetic pigments of different pea morphotypes under changing climatic conditions; 3.To investigate the effects of ozone and UV-B radiation on the biochemi-cal indexes of different pea morphotypes under changing climatic condi-tions. Novelty of the scientific work.the first time the effects of the near-For ground ozone and UV-B radiation on two different pea (Pisum sativumL.) morpho-types (leafy ‘Ilgiai‘ and semi-leafless ‘Profi‘) were investigated under changing cli-mate conditions. The response of these plants to the effects of ozone and UV-B ra-diation was measured. Defended propositions ·The different pea morphotypes respond differently to the effect of anthropogenic factors;
·The morphometric parameters of the different pea morphotypes alters une-qually when responding to the duration and intensity of the investigated factors; ·The different pea morphotypes have unequal photosynthesis mechanisms which determine the protection against photooxidative stress; ·The different pea the systems of soluble sugars and phytohormones diffe-rently are involved in the protection against the effects of ozone and UV-B radiation. Volume and structure of work.The dissertation is composed of the following parts: introduction, literature analysis, research object and methods, research results and discussion, generalization, conclusions, literature (199 sources), list of published works, acknowledgment. The dissertation is illustrated with 11 tables and 33 figures. The volume of the dissertation is 87 pages. Approbation.Work results were published in 7 scientific periodicals including 2 articles in ISI journals and 5 articles referred in International Data Bases. 9 theses were published in conference materials.
RESEARCH OBJECT AND METHODS
Research object.Two varieties of pea (Pisum sativumL.):‘Ilgiai‘ – leafy plants,‘Profi‘–semi-leafless plants. Place and time of research.The experiments were made during the years 2004 – 2008 in Lithuanian University of Agriculture, Laboratory of Agrobiotechnology, Department of Plant Growing and Animal Husbandry and in Lithuanian Institute of Horticulture, Laboratory of Plant Physiology, the complex of phytochambers. The experiments were made when participating in the National scientific project of priority direction “Integrated effect of the anthropogenic changes in climate and environment on the vegetation of forest and agro ecosystems“. Cultivation conditions and investigated factors.During the experiments the peas were cultivated in the substrate of neutral acidity (pH 6 – 6.5). The pea plants were contained in vegetal vessels. 25-30 plants were put in each 5-liter vessel. The experiments were made in three replications. Until germination and one week after germination the plants were grown in the greenhouse and then they
were translocated to the phytochambers. At the end of the experiments morphometric, physiological and biochemical parameters were measured. During the investigation of the ozone and UV-B effects on plants under recent (controlled) climatic conditions the temperature in the phytochambers was maintained at 21oC (day) and 14oC (night). The main forecasted scenarios of the increase of the greenhouse gas emission and the prognostic models of the climate change were followed (IPCC, 2001). During the investigation of the ozone effect on peas, the plants were treated with the following concentrations of ozone: 20 ppb (40 µg m-3) – control; 40 ppb (80 µg m-3); 80ppb (160 µg m-3). Ozone concentrations were generated using ozone monitor OSR – 8 (Ozone Solutions, Inc.) and these concentrations were measured and maintained using ozone monitor OMC – 1108 (Ozone Solutions, Inc.). During the investigation of the UV-B effect on peas, the plants were treated with the following UV-B doses per day: 0 kJ m-2d-1– control; 2 kJ m-2d-1; -4 kJ m-2d1. UV-B radiation was generated using UV-B lamp (TL 40W/12 RS UV-B Medical, Philips). UV-B dose was measured with radiometer VLX – 3 W (Vilber Lourmat). Main investigated morphometric, photosynthetic and biochemical indexes of plants.Morphometric, photosynthetic and biochemical indexes of plants were measured after 1, 3, 5 days and at the end of the experiment (7 days in the case of UV-B radiation and 9 days in the case of ozone). Morphometric parameters: ·Plant height, cm; ·Dry biomass of the above-ground part, mg; ·Leaf area, cm2(WinDias measurer); ·Stomata number, (per area unit) u. Photosynthetic pigments: ·chlorophyllaandb, carotenoids, mg g-1.
Biochemical indexes: ·mono and disaccharides, mg g-1; ·phytohormones, µg g-1. The means and the standard errors were calculated for the statistical analysis. Program package MS EXSEL and ANOVA MINITAB were used. Measurement of photosynthetic pigments.The levels of chlorophylls a, b and carotenoids were measured following the method of Wettstein (Гавриленко 1975). A sample of 0,2 g was taken from the leaves which were picked for the investigation. The sample was ground in a small supply of 100 % acetone and CaCO3. The solution was filtered through a glass filter using a vacuum pump (Vacuum Manifold Accesories). The sample was repeatedly washed with a small supply of acetone until it remained colourless. Then it was poured into a 50-ml flask and supplemented with acetone to the required level. Pigment level was measured in the 100 % acetone extract using spectrophotometer, at the following wavelengths: 440.5 nm – carotenoids; 662 nm – chlorophyll a (Chla); 644 nm – chlorophyll b (Chlb). Measurement of mono- and disaccharides.The method of high pressure liquid chromatography (HPLC) with refractometric detector was used for the separation and measurement of fructose, glucose, sucrose and maltose. A sample of 1–2 g was taken from the green plant mass for the measurement of sugars. The sample was ground and poured over with 4 ml of hot (approximately 75oC) bidistilled water. The sample was extracted for one day when being filtered with filter paper and (just before HPLC analysis) with 0,45 acetate cellulose filter. The precision of the quantitative analysis by the chromatographic method as expressed using relative standard deviation for the peak area is less than 1.8. Detection limit: for fructose 0,01 µg ml-1, for glucose 0,12 µg ml-1, for sucrose 1 and maltose 0,02 µg ml-1. Measurement limit: for fructose 0,03 µg ml-, for glucose 0,27 µg ml-1, for sucrose 0,05 µg ml-1and for maltose 0,02 µg ml-1. Phytohormone measurement.1-g samples of the investigated material (leaves) were powdered in liquid nitrogen and poured over with 10mL propanol-2. Extraction was underway for one day at the temperature of 4oC. Centrifugation (for 5 minutes at 4oof 2500 rpm) was repeated 3 times. Each time theC, at the speed extract was poured off and the sample was poured over with 2 ml propanol-2 afresh. After centrifugation, the extract was evaporated in the rotary evaporator at the 9
temperature of 80oC. The evaporated sample was supplemented with 500 µl 0.1M imidazole buffer (pH 8). The evaporation by the rotary evaporator was continued until the hydrous phase. The sample was diluted to 20 mL with bidistilled water and 0.5 ml 0.1 M imidazole buffer (pH 8). 4 analysed fractions were purified using NH2-columns. Beforehand, NH2-columns were prepared by the sorbent activation with methanol, 5 % acetic acid and bidistilled water. Then the sample was put on the column, the non-analysed substances were washed out with hexane and methanol and the sample was eluted with 5 % acetic acid in 50 % methanol and evaporated using the rotary evaporator. The collected samples were stored at the temperature of 4oC (Wang, 2003). The analyses of gibberellic acid (GA3), indole-3-acetic acid (IAA), abscisic acid (ABA) and zeatin were made using chromatograph ‚Shimadzu HPLC10A‘ (Shimadzu, Japan) with diode-matrix detector (SPD – M 10A VP). The column was termostated at 35oC. The separation was made using column Intersil ODS -2 (150 x 4.6 mm) (Alltech, USA). Mobile phase gradient for the separation of the following compounds: GA3- 40 % methanol with 1 % acetic acid, IAA - 45 % methanol with 1 % acetic acid, zeatin - 50 % methanol with 1 % acetic acid, ABA -55 % methanol with 1 % acetic acid. Detection: 254 nm, 280 nm, 270 nm and 254 nm for GA3, IAA, zeatin and ABA, respectively. Flow rate – 1 ml/min, analysis duration – 10 min.
RESEARCH RESULTS AND DISCUSSION
Ozone effect on the morphometric parameters of different pea morphotypes Ozone effect on plant height and dry biomass When the height response of the different pea morphotypes to ozone was investigated, after the 1-day treatment, higher susceptibility to ozone was already found for the pea (Pisum sativum L.) variety ‘Ilgiai‘, as compared to the variety ‘Profi‘. It was assessed that after the 1-day treatment with ozone concentrations of 80 µg m-3and 160 µg m-3, of the leafy pea variety ‘Ilgiai‘ decreased by 18the height % and 23 %, respectively, as compared to the control plants. As the treatment dura-tion and ozone concentration increased, a tendentious toxic effect of ozone on the increase of the height of the leafy peas ‘Ilgiai‘ was observed. The height of the above-ground part of the semi-leafless peas ‘Profi‘, contrary to the leafy peas, remained stable after the 1-day treatment with 80 µg m-3 and 160 µg m-3concentrations of ozone and was similar to that of the control plants.
A toxic effect of ozone was noted only when the semi-leafless peas were treated with ozone for the longer time (9 days). When the analysis of the susceptibility to ozone effect was made with the different pea morphotypes, it was assessed that after the 1-day treatment with ozone concentrations of 80 µg m-3and 160 µg m-3, the dry biomass of the leafy peas ‘Ilgiai‘ already decreased approximately by 20 %, as compared to the control. At the end of the experiment (after 9 days) the highest tested ozone concentration (160 µg m-3plants to 50 %, as compared) was found to decrease the dry biomass of the leafy to the control variant. The dry biomass of the semi-leafless peas ‘Profi‘, contrary to the leafy peas ‘Ilgiai‘, increased by 13 % after the 1-day treatment with 80 µg m-3ozone. After the 3-day treatment with ozone concentrations of 80 µg m-3and 160 µg m-3, it was assessed that the dry biomass of the semi-leafless peas increased by 49 % and 31 %, respectively, as compared to the control. Only after a longer treatment time, after 5 days and at the end of the experiment (9 days), as ozone concentrations increased, a toxic effect on the dry-biomass accumulation in the semi-leafless peas was observed.
Ozone effect on the leaf area and stomata number When the results of the leaf area measurement were analysed it was assessed that after the 1-day treatment, the leaf area of the leafy peas was already decreased, as compared to the control plants (Fig. 1 A). At the end of the experiment, after the 9-day treatment with ozone concentrations of 80 µg m-3 and 160 µg m-3leaf area of the leafy peas was approximately 50 % smaller., the After the 1-day ozone treatment, no essential differences, as compared to the control plants, were found for the leaf area of the semi-leafless peas. After the 3- day treatment with the highest tested ozone concentration (160 µg m-3), the leaf area of the treated plants was even 35 % larger than that of the control plants. But as the treatment time was prolonged, the leaf area of the treated semi-leafless peas also decreased, as compared to the control plants (Fig. 1 B).