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).

Sujets

• Botany
• Fagus sylvatica
• Quercus robur
• Resting potential

Informations

• Publié par: vilnius_university
• Publié le: 01 janvier 2010
• Nombre de lectures: 30

Extrait

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, H 2 O 2  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 H 2 O 2    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