The signaling role of magnesium protoporphyrin IX and heme in Chlamydomonas reinhardtii [Elektronische Ressource] / vorgelegt von Linda Meinecke

The signaling role of magnesium
protoporphyrin IX and heme in
Chlamydomonas reinhardtii


Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr.rer.nat.)
im Fach Biologie
eingereicht an der



Fakultät für Biologie der
Albert-Ludwigs-Universität Freiburg i.Br.

vorgelegt von Linda Meinecke
Juni 2010 Dekan der Fakultät für Biologie: Prof. Dr. Ad Aertsen
Betreuer der Arbeit: Prof. Dr. C.F. Beck
Koreferent: Prof. Dr. W.R Hess
3. Prüfer: Prof. Dr. G. Radziwill

Promotionsvorsitzender: Prof. Dr. E. Schäfer

Tag der mündlichen Prüfung: 10.11.2010



Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und ausschließlich unter
Verwendung der angegebenen Hilfsmittel angefertigt habe.

Linda Meincke
Freiburg im Breisgau, Juni 2010



Publications

von Gromoff, E.D., Alawady, A., Meinecke, L., Grimm, B. and Beck, C.F. (2008)
Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas.
Plant Cell 20, 552-567.

Meinecke, L., Alawady, A., Schroda, M., Willows, R., Kobayashi, M.C., Niyogi, K.K.,
Grimm, B., and Beck, C.F. (2010)
Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium
protoporphyrin IX.
Plant Mol Biol 72, 643-658.

Manuscript

Björn Voß, Linda Meinecke, Thorsten Kurz, Christoph F. Beck, Wolfgang R. Hess
Hemin and Mg-Protoporphyrin IX Induce Global Changes in Gene Expression in
Chlamydomonas reinhardtii
(eingereicht bei Plant Physiology April, 2010)



































„Der Natur gegenüber zu stehen und seinen Scharfsinn an ihren Rätseln zu erproben, gibt
dem Leben einen ungeahnten Inhalt.“

Alfred Wegener Index 1
1 SUMMARY.......................................................................................................................5
2 INTRODUCTION .........................................................7
2.1 Tetrapyrrole biosynthesis in photosynthetic organisms ...................................................................... 7
2.2 Regulation of tetrapyrrole biosynthesis .............................................................................................. 11
2.3 Interorganellar communication ........................................................ 19
2.4 Involvement of tetrapyrroles in interorganellar communication ..................................................... 23
2.4.1 Interorganellar signaling from the mitochondria to the nucleus: heme as a mitochondrial signal ... 23
2.4.2 Interorganellar signaling from the chloroplast to the nucleus: heme and Mg-porphyrins as
plastidal signals ............................................................................................................................ 24
2.4.2.1 Role of Mg-porphyrins in chloroplast-to-nucleus signaling in higher plants............................ 24
2.4.2.2 Role of heme and Mg-porphyrins in chloroplast-to-nucleus signaling in Chlamydomonas ..... 28
2.5 Chlamydomonas reinhardtii as a plant model organism..................................................................... 35
2.6 Aim of this work.................................................................................................................................... 39
3 MATERIALS AND METHODS...................................................................................40
3.1 Chemicals, Enzymes, Kits and Ladders ........................................... 40
3.1.1 Enzymes ....................................................................................................................................... 40
3.1.2 Ladders ...................................................................................... 40
3.1.2.1 DNA Ladder .......................................................................... 40
3.1.2.2 RNA Ladder........................................................................... 40
3.1.2.3 Protein Ladder ........................................................................................ 40
3.2 DNA digestion, gel electrophoresis and purfication of DNA fragments........................................... 41
3.2.1 DNA digestion with restriction endonucleases.......................... 41
3.2.2 Native DNA gel electrophoresis (Sambrook, 1989)..................................................................... 41
3.2.3 Gel purification of DNA fragments........................................... 41
3.3 Buffers and Solutions............................................................................................................................ 42
3.3.1 Frequently used buffers and solutions ....................................... 42
3.3.2 Solutions and media for the culture of Chlamydomonas reinhardtii............................................ 42
3.3.2.1 Stock solutions for Tris-acetate-phosphate medium (TAP) and Tris-minimal-phosphate
medium (TMP) ......................................................................................................................... 42
3.3.2.2 TAP and TMP medium.......................................................... 43
3.3.3 Media for the culture of bacteria ............................................... 44
1 Index 2
3.4 Strains and methods used for Chlamydomonas .................................................................................. 45
3.4.1 Wild type and mutant strains of Chlamydomonas reinhardtii...................................................... 45
3.4.2 Growth of Chlamydomonas reinhardtii cell culture...................................... 46
3.4.3 Heat shock ................................................................................. 46
3.4.4 Cryoconservation of algal cells .................................................................................................... 46
3.4.5 Determination of phenotypes after growth under different light conditions ................................ 47
3.4.6 Gametogenesis and crossing of Chlamydomonas reinhardtii strains (based on the procedure
developed by (Levine and Ebersold, 1960) ............................... 47
3.4.7 Random spore analysis................................................................................................................. 48
3.4.8 Autolysin preparation (Harris, 1989)......................................... 48
3.4.9 Removal of cell walls ................................................................ 48
3.4.10 Transformation of Chlamydomonas reinhardtii........................ 48
3.4.11 Isolation of Chlamydomonas reinhardtii genomic DNA and Southern blot analysis .................. 49
3.4.11.1 Isolation of Chlamydomonas reinhardtii genomic DNA using the CTAB-method..................49
3.4.11.2 Southern blot analysis............................................................................................................... 50
3.4.12 Radioactive DNA labeling with random primer for Southern and Northern blot analyses .......... 52
3.4.13 Radioactive labeling of transcript probes .................................. 52
3.4.14 Polymerase chain reaction (PCR).............................................. 53
3.4.15 Isolation of Chlamydomonas reinhardtii total RNA .................................................................... 53
3.4.16 Denatured formaldehyde gel electrophoresis of RNA.................................................................. 54
3.4.17 Northern blot analysis ............................................................... 55
3.4.17.1 Northern transfer.................................. 55
3.4.17.2 Northern hybridization.............................................................................................................. 56
3.4.17.3 Probes used for hybridization ................................................ 57
3.4.18 Optimization of the protocol for Northern hybridization with transcript probes.......................... 58
3.4.19 Quantification of hybridization signals ..................................... 59
3.4.20 Spectrophotometric determination of chlorophyll concentration ................................................. 60
3.4.21 Determination of porphyrin steady state levels ............................................................................ 60
3.4.22 Sources of porphyrins and feeding experiments for the microarray............................................. 61
3.4.23 Isolation of total protein from Chlamydomonas reinhardtii...... 61
3.4.23.1 Determination of protein concentration with amido black........................................................ 61
3.4.24 SDS-Page (polyacrylamide gel electrophoresis) .......................................................................... 62
3.4.25 Western blot .............................................................................. 64
3.4.26 Immunological detection of proteins with antibodies................ 64
3.4.26.1 Antibodies...............................................