Analysis of proteins involved in chlorophyll catabolism [Elektronische Ressource] : the impact of chlorophyllase and water soluble chlorophyll protein on the reduction of undesirable pigments in crop plants / Sridevi Damaraju. Gutachter: Bernhard Grimm ; Heiko Lockstein ; Kurt Zoglauer

Srk

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Analysis of Proteins Involved in Chlorophyll Catabolism:
The impact of Chlorophyllase and Water Soluble Chlorophyll Protein on
the reduction of undesirable pigments in crop plants


DISSERTATION


zur Erlangung des akademischen Grades
doctor rerum naturalis
(Dr. rer. nat.)
im Fach Biologie


eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin


von
Master of Genetics, Sridevi Damaraju




Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies


Dekan der Mathematisch Naturwissenschaftliche Fakultät I
Prof. Dr. Lutz-Helmut Schön


Gutachter
Prof. Dr. Bernhard Grimm
PD. Dr. Heiko Lockstein
PD. Dr. Kurt Zoglauer


Tag der mündlichen Prüfung: 11- June-2010
TABLE OF CONTENTS
ABBREVIATIONS ............................................................................................................... 1
ZUSAMMENFASSUNG ...... 3
ABSTRACT .......................... 5

1 INTRODUCTION ......................................................................................................... 7
1.1 Chlorophyll – the Green Power ................. 7
1.2 Green seed problem ... 8
1.3 Minimising the Green Seed problem ......... 9
1.4 Chlorophyll catabolism ........................................................................................... 10
1.4.1 Chlorophyllase ................................. 13
1.4.1.1 Localization of Chlase in the chloroplasts ................. 13
1.4.1.2 Enzymatic kinetics of Chlase .................................... 14
1.4.1.3 Molecular properties of the genes encoding Chlase in different plant
species ........................................................................ 18
1.4.1.4 Post translational regulation of Chlase expression and activity ................ 24
1.4.1.5 Functional roles of Chlase in plants .......................................................... 24
1.4.2 Significance of others enzymes in Chl catabolism .......... 24
1.4.3 Water Soluble Chlorophyll Protein (WSCP) ................... 26
1.4.3.1 Classification of WSCPs ........................................................................... 26
1.4.3.1.1 Class I WSCPs ..................... 26
1.4.3.1.2 Class II WSCPs ................... 27
1.4.3.2 Molecular and functional aspects of WSCPs ............ 28
1.4.3.3 Pigment binding and photoprotective function of WSCPs ........................ 30
1.4.3.4 Functions of WSCPs in plant system ................................ 34

2 AIM ............................................................................................. 35

3 MATERIALS .............................................................................................................. 37
3.1 Plasmid DNA ........... 37
3.2 Oligonucleotides ...... 37
3.3 Organisms ................ 37
3.4 Antibodies ................................................................................................ 37
3.5 Chemicals and material ........................... 38
3.6 Equipments .............. 39
3.7 Glass and Plastic ware ............................................................................................. 39
3.8 Kits, Substances, Enzymes and Buffers .................................. 39
3.9 Solutions and buffers ............................... 40

4 METHODS .................................................................................................................. 46
4.1 Reverse transcription 46
4.2 Polymerase chain reaction ....................... 46
4.3 Overexpression of Chlase in E. coli ........................................................................ 46
4.4 Protein purification and antibody production .......................... 47
4.5 Agarose gel electrophoresis ..................... 47
4.6 SDS-PAGE analysis ................................ 48
4.7 Western blot Analysis .............................................................. 48
4.8 In vitro enzyme activity assays ................................................ 48
4.8.1 Extraction of Chl for enzyme assays ............................... 49
4.8.2 Conversion of Chl to Phe ................................................ 49
4.8.3 Enzyme assays of Chlase ................. 50
i
4.8.4 Coupled assays with Chlase and WSCP .......................................................... 53
4.9 Production and analysis of transgenic tobacco plants ............. 53
4.9.1 Propagation of sterile tobacco plants ............................... 53
4.9.2 Transformation of tobacco plants .................................................................... 54
4.10 Plant growth and treatment regimes ........ 55
4.11 Analysis of transgenic tobacco plants ..... 55
4.11.1 Genomic DNA isolation from plants ............................... 55
4.11.2 Initial screening of plants by PCR ................................................................... 56
4.11.3 Total RNA isolation from plants ..... 56
4.11.4 Radioactive labelling of probes ....... 56
4.11.5 Southern blot analysis ...................................................................................... 57
4.11.6 Northern blot analysis 57
4.11.7 Protein extraction from plants ......... 58
4.11.7.1 From Chlase overexpressor plants ............................. 58
4.11.7.2 From WSCP overexpressor plants ................................ 58
4.11.8 Selection of Chlase overexpressor plants based on the functional activity
of protein in vitro ................................ 59
4.11.9 Isolation and fractionation of chloroplasts from Chlase overexpressor
plants ................................................................................ 59
4.11.10 Experiments on Chlase overexpressor plants .................. 60
4.11.11 Experiments on WSCP overexpressor plants .................. 60
4.11.12 Analysis of pigments from transgenic tobacco plants ..................................... 61
4.11.12.1 Estimation of steady state levels of Chl and Chlide a ............................... 61
4.11.12.2 NCC extraction and quantification ............................................................ 62
4.11.12.3 Determination of Pchlide Content ............................. 62
4.11.12.4 Determination Chl catabolite levels in Chlase overexpressor plants
due to in vitro activity of recombinant CcChlase ...................................... 62
4.11.13 Determination of ALA-synthesizing capacity ................. 63
4.11.14 Chl fluorescence experiments of WSCP overexpressor plants ....................... 63
4.11.15 Estimation of peroxidase activity in WSCP overexpressor plants .................. 64
4.11.16 Statistical analysis ........................................................................................... 65

5 RESULTS .................................................... 66
5.1 Chlorophyllase ......................................................................... 66
5.1.1 CcCHLASE (Chlase gene from Citrus clementii) as transgene ....................... 66
5.1.2 Purification of His-CcChlase for production of antiserum against Chlase ..... 66
5.1.3 In vitro enzyme activity assays of recombinant His-CcChlase protein ........... 68
5.1.3.1 Enzymatic activity of His-CcChlase protein ............................................. 69
5.1.3.2 Optimization of temperature and pH for His-CcChlase activity ............... 72
5.1.3.3 Effect of reaction time on enzyme activity of His-CcChlase .................... 73
5.1.3.4 Effect of acetone concentration on His-CcChlase activity ........................ 74
5.1.3.5 Effect of substrate concentration on His-CcChlase activity ...................... 75
5.1.3.6 Effect of protein concentration on His-CcChlase activity ......................... 75
5.1.3.7 Substrate specificity of His-CcChlase ....................................................... 76
5.1.3.8 Effect of phytol concentration on His-CcChlase activity .......................... 78
5.1.3.9 His-CcChlase activity on thylakoid membranes ....................................... 79
5.1.3.10 Effect of various metal ions on His-CcChlase activity .............................. 79
5.1.3.11 Effect of reagents that modify the functional groups on
His-CcChlase activity ................................................................................ 80
5.1.3.12 Effect of oil concentration on His-CcChlase activity ................................ 81
ii
5.1.3.13 Optimization of different extraction solutions in the oil assays for
maximal Chlide recovery........................................................................... 81
5.1.3.14 Activity of His-CcChlase on Pheophytin as substrate ............................... 83
5.1.3.15 Activiis-CcChlase on Chl in oils at different stages of
oil refining ................................................................................................. 84
5.1.4 Tobacco as a model for development of transgenic plants .............................. 85
5.1.5 Production and preliminary analysis of transgenic tobacco plants
overexpressing CcCHLASE ...........................................