Accumulation and biological activity of oxidized lipids in Anabaena PCC 7120 [Elektronische Ressource] / vorgelegt von Pham Phuoc Nhan

Accumulation and Biological Activity of
Oxidized Lipids in Anabaena PCC 7120




Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg





vorgelegt von
Pham Phuoc Nhan
aus Cantho, Vietnam





Würzburg, 2007
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Eingereicht am:
Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr.
1. Gutachter: Prof. Dr. Martin J. Müller
2. Gutachter: Prof. Dr. Werner Kaiser

Tag des Promotionskolloquiums:
Doktorurkunde ausgehändigt am:









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„Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes“























3Table of contents
I. INTRODUCTION................................................................................................................... 1
I.1 General introduction into the cyanobacteria ........... 1
I.1.1 The appearance of cyanobacteria .................................................................................... 1
I.1.2 Cyanobacteria as architects of the early earth atmosphere ............. 1
I.1.3 Diversity, adaptation and ecological roles of cyanobacteria........................................... 3
I.1.4 Toxicity from cyanobacteria ........................................................................................... 4
I.1.5 Some cyanobacterial species are biological model organisms
for scientific research ...................................................................................................... 4
I.1.6 Cyanobacterial fatty acid profile and its meaning........................... 5
I.2 Oxidative stress in cyanobacteria ............................................................................................ 6
I.3 Phytoprostanes ........................................................ 9
I.3.1 Prostaglandins and isoprostanes in animals .................................... 10
I.3.2 Jasmonates and phytoprotanes in plants ......................................... 12
I.3.2.1 Jasmonates............................................................................... 12
I.3.2.2 Discovery and biosynthesis of phytoprostanes in plants......... 14
I.3.2.3 Occurrence, analysis and classification of phytoprostanes in plants ...................... 16
I.3.2.4 Biological functions of phytoprostanes................................................................... 19
I.4 Aims of the work..................................................................................... 20
II. MATERIALS AND METHODS.......................................................... 23
II.1 Chemicals ............................................................................................... 23
II.2 Materials ................................................................ 23
II.3 Equipments ............................ 23
II.4 Cell culture of Anabaena PCC 7120 und Synechocystis PCC 6803...... 26
II.5 Determination of dry weights of cell suspension by optical density ..................................... 27
II.6 Solid phase extraction (SPE) ................................................................. 28
II.7 Hydrogenation, methoximation, and derivatisation of oxylipins .......... 28
II.7.1 Hydrogenation ............................................................................................................... 28
II.7.2 Methylation of carboxyl group by trimethylsilyl-diazomethane ... 29
II.7.3 Methoximation of keto group by methoxyamine .......................................................... 29
II.7.4 Esterification of carboxyl groups by pentafluorobenzylbromide (PFB-Br) .................. 30
II.7.5 Preparation of trimethylsilyl ether derivatives with N,O-Bis-
4 (trimethylsilyl)trifluoracetamide (BSTFA) ................................................................... 30
II.8 Formation of phytoprostanes type III and IV by autoxidation of g-linolenic acid ................ 31
II.8.1 Hydrolysis of Borage oil ................................ 31
II.8.2 Autoxidation of g-linolenic acid and quantification of PPF type III and IV ................ 31 1
II.8.3 Autoxidation of g-linolenic acid and quantification of PPE type III and IV by 1
isomerisation to corresponding PPB type III and IV ................................................... 31 1
II.8.4 Formation and isolation of PPA type III and IV by acidic dehydration of 1
PPE type III and IV ...................................................................................................... 33 1
II.9 Fatty acid quantification in Anabaena und Synechocystis ..................................................... 34
II.10 Analysis of total hydroxy fatty acid in Anabaena by GC-MS............. 35
II.11 Determination of phytoprostanes from and Synechocystis by GC-MS .............. 35
II.11.1 Extraction of free PPF from cells and medium by GC-MS........................................ 35 1
II.11.2 Analysis of free PPE -MS .......... 36 1
II.11.3 Analysis of total PPF and PPE from the cells by GC-MS ........ 36 1 1
II.12 Determination of free and total PPF and PPE in Synechocystis by HPLC-MS/MS ......... 37 1 1
II.13 Analysis of PPF and PPE in Anabaena grown under high light intensity and 1 1
oxidative stress .................................................................................................................... 38
II.14 Investigation of protective effects of oxylipins on Anabaena under
oxidative stress condition .................................................................................................... 38
II.15 Analysis of H O and PPF effects on isiA (iron stress induced protein A) 2 2 1
expression in Anabaena....... 39
II.15.1 RNA isolation from Anabaena .................................................................................... 40
II.15.2 Reverse Transcription – Polymerase Chain Reaction (RT-PCR) ................................ 40
II.16 Analysis of PPF effect on the Anabaena proteome ............................................................ 41 1
II.16.1 Protein extraction in Anabaena cells ........................................................................... 41
II.16.2 Determination of Anabaena proteins by 1D-gelelectrophoresis.. 42
II.16.3 Liquid isoelectric focusing........................................................................................... 43
II.16.4 Protein separation by SDS-PAGE ............... 44
II.16.5 Two-dimensional gel electrophoresis according to Görg et al. (2004) ....................... 44
II.16.6 In-gel digestion of protein spots .................................................................................. 45
II.16.7 Protein analysis by nano-LC-MS/MS .......... 45
II.17 Shinorine analysis in Anabaena after ultra violet light (UV light), copper sulphate,
jasmonate and phytoprostane treatment .............................................................................. 46
5II.18 Analysis of tocopherols in Anabaena after ultra violet light (UV light),
copper sulphate, jasmonate and phytoprostanes treatment .................................................. 46
II.19 Salicylic acid, jasmonate, and phytohormone analysis in Anabaena .................................. 46
II.20 Investigation of protective effects of PPF on Pseudomonas syringae and Escherichia 1
coli under oxidative stress condition ................................................................................... 47
II.21 Analysis of free PPF and PPE in hay by GC-MS ............................. 48 1 1
II.22 HPLC methods..................................................... 48
II.22.1 HPLC parameters for MAA analysis ........................................................................... 48
II.22.2 HPLC parameters for tocopherols analysis . 49
II.22.3 HPLC method for PPB and PPA analysis................................................................. 49 1 1
II.23 GC-MS methods .................................................................................................................. 50
II.24 HPLC-MS/MS methods....... 51
II.24.1 HPLC-MS/MS setting for PPF ................... 51 1
II.24.2 HP LC-MS/MS setting for phytohormones .................................................................. 52
II.24.3 HPLC-MS/MS setting for PPB .................................................................................. 53 1
II.25 Nano-HPLC-MS/MS method for proteome analysis........................... 54
III. RESULTS .......................