Nitellopsis obtusa (desv.) J. Groves cell response to allochtonous stressors of fagus sylvatica l. And quercus robur l. Leaf litter extracts ; Nitellopsis obtusa (Desv.) J. Groves ląstelių atsakas į alochtoninius stresorius – Fagus sylvatica L. ir Quercus robur L. lapų paklotės ekstraktus

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VILNIUS UNIVERSITY INSTITUTE OF BOTANY OF NATURE RESEARCH CENTRE Reda Grigutytė NITELLOPSIS OBTUSA (DESV.) J. GROVES CELL RESPONSE TO ALLOCHTONOUS STRESSORS OF FAGUS SYLVATICA L. AND QUERCUS ROBUR L. LEAF LITTER EXTRACTS Doctoral Dissertation Biomedical sciences, botany (04 B) Vilnius, 2010 Dissertation was performed in 2005–2009 at the Institute of Botany of Nature Research Centre (Lithuania) and Leibniz–Institute of Freshwater Ecology and Inland Fisheries (Germany). Scientific supervisor – Dr. Levonas Manusadžianas (Institute of Botany of Nature Research Centre, biomedical sciences, biophysics – 02 B) Scientific advisor – PD. Dr. Stephan Pflugmacher (Leibniz–Institute of Freshwater Ecology and Inland Fisheries, biomedical sciences, biology – 01 B) 2 VILNIAUS UNIVERSITETAS GAMTOS TYRIMŲ CENTRO BOTANIKOS INSTITUTAS Reda Grigutytė NITELLOPSIS OBTUSA (DESV.) J. GROVES LĄSTELIŲ ATSAKAS Į ALOCHTONINIUS STRESORIUS – FAGUS SYLVATICA L. IR QUERCUS ROBUR L. LAPŲ PAKLOTĖS EKSTRAKTUS Daktaro disertacija Biomedicinos mokslai, botanika (04 B) Vilnius, 2010 3 Disertacija buvo rengiama 2005–2009 metais Gamtos tyrimų centro Botanikos institute ir Leibnico gėlųjų vandenų ir gėlavandenių žuvų ekologijos institute. Mokslinis vadovas – Dr. Levonas Manusadžianas (Gamtos tyrimų centro Botanikos institutas, biomedicinos mokslai, biofizika – 02 B).

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VILNIUS UNIVERSITY INSTITUTE OF BOTANY OF NATURE RESEARCH CENTRE         Reda Grigutytė  
 NITELLOPSIS OBTUSA(DESV.) J. GROVES CELL RESPONSE TO ALLOCHTONOUS STRESSORS OFFAGUS SYLVATICAL. AND QUERCUS ROBURL. LEAF LITTER EXTRACTS    Doctoral Dissertation Biomedical sciences, botany (04 B)         Vilnius, 2010
Dissertation was performed in 2005–2009 at the Institute of Botany of Nature
Research Centre (Lithuania) and Leibniz–Institute of Freshwater Ecology and Inland Fisheries (Germany).  
Scientific supervisor – Dr. Levonas Manusadžianas (Institute of Botany of Nature Research Centre, biomedical sciences, biophysics – 02 B)
 Scientific
advisor
 
PD. Dr. Stephan Pflugmacher
(Leibniz–Institute
of
Freshwater Ecology and Inland Fisheries, biomedical sciences, biology – 01 B)  
 
2
 
 
VILNIAUS UNIVERSITETAS GAMTOS TYRIMŲ CENTRO BOTANIKOS INSTITUTAS         Reda Grigutytė   NITELLOPSIS OBTUSA(DESV.) J. GROVES LĄSTELIŲ ATSAKAS Į ALOCHTONINIUS STRESORIUS –FAGUS SYLVATICAL. IR QUERCUS ROBURL. LAPŲ PAKLOTĖS EKSTRAKTUS    Daktaro disertacija Biomedicinos mokslai, botanika (04 B)         Vilnius, 2010
Disertacija buvo rengiama 2005–2009 metais Gamtos tyrimų centro Botanikos
institute ir Leibnico gėlųjų vandenų ir gėlavandenių žuvų ekologijos institute.  Mokslinis vadovas – Dr. Levonas Manusadžianas (Gamtos tyrimų centro
Botanikos institutas, biomedicinos mokslai, biofizika – 02 B).   
Mokslinis konsultantas – PD. Dr. Stephan Pflugmacher (Leibnico gėlųjų vandenų ir gėlavandenių žuvų ekologijos institutas, biomedicinos mokslai,
biologija – 01 B).
    
 
                 
4
TABLE OF CONTENTS
List of abbreviations .......................................................................................... 8 Introduction .................................................................................................... 10 1.  15Literature overview ................................................................................ 1.1.  matter ............................ 15Some egzogenic sources of natural organic 1.1.1. F. sylvatica 16leaf litter .................................................................. 1.1.2. Q. roburleaf litter....................................................................... 17 1.2. Freshwater charophyte,N. obtusa..................................................... 18 1.3. Reactive oxygen species ................................................................... 19 1.4.  in plants ............................ 22Oxidative stress and defense mechanis m 1.4.1.  2.5.1.18)Glutathione S-transferase (GST: EC 24 ............................ 1.4.2. Catalase (CAT: EC 1.11.1. ...................................................... 26 6) 1.4.3. Glutathione reductase (GR: EC 1.8.1. 7)................................ ...... 27 1.4.4. Glutathione peroxidase (GPx: EC 1.11. 1.9) ................................ 27 1.4.5.  ................................. 27 1.15.1.1)Superoxide dismutase (SOD: EC 
1.5.  peroxidation .... lipidHydrogenperoxide, total antioxidant status and 28 
1.6. Electrophysiological reaction ofN. obtusacells................................ 30 
1.7.  32Lethality, as a measure of toxicity..................................................... 2. Methods.................................................................................................. 33 
 
2.1. 
2.2. 
2.3. 
2.4. 
2.5. 
2.6. 
Plant material .................................................................................... 33 
Preparation ofQ. roburandF. sylvaticaleaf extracts ....................... 33 Leaf litter degradation extracts (L LDEs) ........................................... 34 
Chemical characterization of leaf extracts and Lake water ................ 34 
Dose- and time-dependent exposure ofN. obtusatoQ. robur and
F. sylvatica 35leaf extracts................................................................... 
Exposure of charophyte cells  ............................................ 36to LLDEs
2.7. Protein extraction .............................................................................. 36
2.8. Measurement of enzymatic acti vities ................................................ 37
2.8.1. 
2.8.2. 
2.8.3. 
 
 
 
Catalase ...................................................................................... 37 Glutathione S-transferase ............................................................ 37 
Glutathione reductase ................................................................. 38 
5
2.8.4.  39Glutathione peroxidase ............................................................... 2.8.5. Calculation of specific enzymatic activity by photometric measurement ............................................................................... 39 2.8.6. Superoxiddismutase .................................................................... 40 2.8.7.  drogenMeasurement of cell internal hy peroxide......................... 41 2.8.8. Measurement of the total antioxidant status ................................ 42 2.8.9.  .............................................Measurement of lipid peroxidation 42 2.9.  43Electrophysiological experiments ..................................................... 2.10. Cell Lethality testing ......................................................................... 45 2.11.  45Statistical analysis............................................................................. 3. Results.................................................................................................... 46 3.1. Enzymatic activity, H2O2 content, total antioxidant content and lipid peroxidation inN. obtusa................ .64................................ ................ 3.1.1. Dose – response effects induced byF. sylvaticaandQ. roburleaf extracts ....................................................................................... 46 3.1.1.1. Microsomal glutathione S-transferase assayed with the
 
3.1.1.2. 
3.1.1.3. 
3.1.1.4. 
3.1.1.5. 
3.1.1.6. 
3.1.1.7. 
conjugating substrate 1-chloro-2,4 -dintrobenzene (CDNB) .... 46 
Cytosolic glutathione S-transferase assayed with the
conjugating substrate CDNB.................................................. 47 Catalase ................................................................................. 48 
Glutathione reductase ............................................................ 49 
Glutathione peroxidase .......................................................... 50 
Superoxide dismutase ............................................................ 51
Hydrogen peroxide content .................................................... 52
 
 
3.1.1.8. Total antioxidant content ....................................................... 53 3.1.1.9. Lipid peroxidation ................................................................. 53 3.1.2. Time-dependent effects induced byF. sylvatica andQ. robur leaf
extracts ........................................................................................ 54 
3.1.2.1. 
3.1.2.2. 
3.1.2.3. 
Microsomal glutathione S-transferase, measured with CDNB 54 Cytosolic glutathione S-transferase, m easured with CDNB .... 55 
Catalase ................................................................................. 57 
6
 
 
Research articles and congress presentations ................................................... 92
References ...................................................................................................... 78
 
 
 
 
7
 
 
3.1.3.3. 
Glutathione reductase ............................................................ 62
3.1.3.4. 
Hydrogen peroxide content .................................................... 63
3.1.3.5. 
The electrophysiological response ofN. obtusa affected by cells
3.2. 
F. sylvaticaandQ. robur 64leaf extracts ............................................. 
The lethality response ofN. obtusa exposed to cellsF. sylvatica and
3.3. 
Q. robur ....................................................................... 65leaf extracts 
Discussion .............................................................................................. 67
4. 
 
Conclusions .................................................................................................... 75 
Acknowledgements ......................................................................................... 77
3.1.3. Effects induced by 30-day-decomposing of leaf litter extracts
Glutathione reductase ............................................................ 58 
fromF. sylvaticaandQ. robur.................................................... 59 
3.1.2.4. 
3.1.3.1. 
Dissolved organic carbon content .......................................... 59 
3.1.3.2. 
Cytosolic glutathione S-transferase, assayed with the
Catalase ................................................................................. 61
conjugating substrate CDNB.................................................. 60
freshwater of variance pond water e peroxidase lvaticaleaf extract 2,4-dinitrobenzene GST onucleic acid organic carbon thritol etriaminepentaacetic acid
List of abbreviations  AFW Artificial ANOVA Analysis APW Artificial APX Ascorbat BLEFagus sy CAT Catalase CDNB 1-chloro-cGST Cytosolic DNA Deoxyrib DOC Dissolved DTE Dithioery DTPA Diethylen DW Dry weig EDTA Ethylene g Gram GAPDH Glycerald GPx Glutathio GR Glutathio GSH GSSG GST H2O2  HNE HS L LLDEs LPO MDA mg
 
ht diaminetetracetic acid ehyde-3-phosphate dehydrogenase ne peroxidase ne reductase
Reduced glutathione Glutathione disulphide Glutathione S-transferase Hydrogen peroxide Hydroxynonenal Humic substances Litre Leaf litter degradation extracts Lipid peroxidation Malondialdehyde Milligram
8
mGST 
mL NADPH NAP-5
nkat NOM
OLE RNA
ROS SOD TOSC
μL
 
 
 
Membrane bound GST
Millilitre Reduced nicotinamide adenine dinucleotide phosphate Sephadex G-25 Column
Nano katal Natural organic matter
Quercus roburleaf extract Ribonucleic acid
Reactive oxygen species Superoxide dismutase Total antioxidant status
Microlitre
9