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

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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.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 34
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Source : D-NB.INFO/1011062674/34
Nombre de pages : 195
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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................................................................................................................................. 65
3.4.27 Determination of ALA synthesizing capacity (in cooperation with A. Alawady, Berlin)............ 66
3.4.28 Determination of enzyme activities........................................... 66
3.5 The Chlamydomonas reinhardtii expression microarray ................................................................... 66
3.5.1 Microarray design (done by W. Hess, Björn Voss and Thorsten Kurz) ....................................... 66
3.5.2 Generation of RNA samples for the microarray experiments ...................................................... 67
3.5.3 Hybridization of the microarray (done by Thorsten Kurz, ZBSA Freiburg) ................................ 68
2 Index 3
3.6 Strains and methods used for bacteria................................................................................................ 68
3.6.1 Escherichia coli (E. coli) strain .................................................................................................... 68
3.6.2 Plasmids .................................................................................... 69
3.6.3 BAC library screening by hybridization with a CHLM probe...................................................... 69
3.6.3.1 Other BAC clones used for the identification of mutation sites................................................ 71
3.6.4 Culturing of bacteria..................................................................................................................... 71
3.6.5 Generation of transformation-competent bacteria and transformation of bacteria ....................... 72
3.6.6 Preparation of plasmid DNA (Sambrook, 1989) ....................... 73
3.6.6.1 Maxi preparation of plasmid DNA ........................................................................................... 73
3.6.6.2 Mini preparation of plasmid DNA..................... 74
3.6.7 Preparation of cosmid DNA ...................................................... 74
4 CHARACTERIZATION OF CHLAMYDOMONAS REINHARDTII MUTANTS
WITH DEFECTS IN CHLOROPHYLL BIOSYNTHESIS: DETERMINATION OF
PHENOTYPE, BIOCHEMICAL AND GENETIC ANALYSES ......................................75
4.1 Results .................................................................................................................................................... 75
4.1.1 Biochemical analyses of mutants defective in chlorophyll biosynthesis: Determination of the
content of chlorophyll precursors in dark-grown cultures............................................................ 75
4.1.2 Determination of phenotypes of mutants defective in chlorophyll biosynthesis growing under
different light conditions .............................................................................................................. 78
4.1.3 Genetic analyses of mutants defective in chlorophyll biosynthesis: Are the mutant phenotypes
caused by single or possibly multiple mutations? ..................... 80
4.1.4 Identification of the mutated genes by complementation using defined clones from a
Chlamydomonas BAC library ...................................................................................................... 81
4.1.5 Analysis of the gene expression of selected genes in the mutants after a dark-to-light shift........ 83
4.1.5.1 RNA blot analyses of light induction of nuclear genes HEMA, CHLH and HSP70A after a
dark-to-light shift ...................................................................................................................... 83
4.1.5.2 RNA blot analyses of light induction of nuclear genes LHCB-I, LHCII-3 and LHCII-4.......... 85
4.1.5.3 Is the mutation responsible for the lack of chlorophyll also responsible for the deregulation
of genes?..................... ........................................................... 86
4.2 Discussion............................................................................................................................................... 87
5 HEME, A PLASTID-DERIVED REGULATOR OF NUCLEAR GENE
EXPRESSION IN CHLAMYDOMONAS..........................90
6 CHLOROPHYLL-DEFICIENT MUTANTS OF CHLAMYDOMONAS
REINHARDTII THAT ACCUMULATE MAGNESIUM PROTOPORPHYRIN IX .....91
3 Index 4
7 EFFECT OF MUTATIONS IN GENES FOR MG-CHELATASE AND
MG-PROTOPORPHYRIN IX METHYLTRANSFERASE ON GUN4 PROTEIN........92
7.1 Results .................................................................................................................................................... 92
7.1.1 GUN4 protein but not GUN4 mRNA is missing in mutants defective in CHLM......................... 92
7.1.2 MgPMT and GUN4 protein levels after complementation with wild-type CHLM in mutants
chlM-1 and chlM-2................. 93
7.1.3 GUN4 protein levels in Mg-chelatase mutants............................................................................. 94
7.1.4 Light-regulation of GUN4 in wild type and chlM mutants........................................................... 94
7.1.5 Effect of the absence of GUN4 on the activity of enzymes crucial for tetrapyrrole biosynthesis.....95
7.1.6 GUN4 protein levels in wild type exposed to high light intensities ............................................. 97
7.2 Discussion............................................................................................................................................... 98
8 HEMIN AND MGPROTO INDUCE GLOBAL CHANGES IN GENE
EXPRESSION IN CHLAMYDOMONAS REINHARDTII................................................103
8.1 Results .................................................................................................................................................. 104
8.1.1 Experimental set-up................................................................. 104
8.1.2 The expression of about 800 genes is affected ........................ 105
8.1.3 Control for the validity of array-generated data ......................................................................... 106
8.1.4 Time course of changes in mRNA levels observed upon feeding of MgProto or hemin ........... 106
8.1.5 Four regulatory groups of genes that differ in their response to MgProto and hemin ................ 109
8.1.6 Comparison of microarray data with Northern blot data for selected genes .............................. 110
8.1.7 Are there PREs in the promoters of MgProto and hemin induced genes?.................................. 115
8.1.8 The function of genes regulated by MgProto and hemin............................................................ 117
8.1.9 Global changes in the transcriptome in response to heat shock.................................................. 120
8.1.10 Overlap in genes responding to tetrapyrroles and heat shock..................................................... 120
8.2 Discussion............................................................................................................................................. 121
9 SUMMARIZING DISCUSSION ................................................................................128
10 ABBREVIATIONS....................................................138
11 REFERENCES .............................................................................................................140


4 1. Summary 5
1 Summary
Chlamydomonas reinhardtii harbors, like al other eukaryotic algae and plants, three
DNA-containing organelles. The nucleus exerts a tight control over gene expression in these
organelles (Goldschmidt-Clermont, 1998; Leon et al., 1998; Hess and Börner, 1999), whereas
plastids and mitochondria generate retrograde signals that in turn regulate the expression of
nuclear genes. In C. reinhardtii a tetrapyrrole-derived signaling pathway was elabo rated
based on the observation that feeding of Mg-protoporphyrin IX (MgProto) and
Mg-protoporphyrin IX monomethyl ester (MgProtoMe) in the dark induce the chaperone
genes HSP70A and HSP70B. Those genes are also induced by shifting cell cultures from dark
to light (Kropat et al., 1997). A transient light-induced increase in MgProto and/or
MgProtoMe appears to be a prerequisite for the activation of these genes by light (Kropat et
al., 2000). However, since an accumulation of these tetrapyrroles in dark-grown cells did not
activate gene expression, it was postulated that light in addition is required to make the
plastid-produced tetrapyrroles accessible to downstream signal transduction components in
the cytosol and nucleus (Kropat et al., 2000; Beck, 2005; Meinecke et al., 2010).
From a collection of 40 Chlamydomonas reinhardtii mutants defective in chlorophyl
biosynthesis, 12 chlH and 5 chlD mutants in the Mg-chelatase that catalyzes the insertion of
magnesium into protoporphyrin IX were identified. The brown, light-sensitive mutants
showed reduced levels of Mg-tetrapyrroles. Four Mg-chelatase mutants (chlH-1, chlH-2,
chlD-1, chlD-2) were characterized further (von Gromoff et al., 2008). All four lack
chlorophyll, show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In
the mutants, light induction of HSP70A was preserved, although MgProto has been
implicated in this induction. Hemin feeding to cultures in the dark induced HSP70A. This
induction was mediated by the same plastid response element (PRE) in the HSP70A promoter
that has been shown to mediate induction by MgProto and light. Other nuclear genes that
harbor a PRE in their promoters were also inducible by heme. A hallmark of this regulation is
the transient activation of genes after treatment with MgProto, hemin, or light. One
mechanism ensuring only transient gene activation appears to be based on the shut-down of
the signaling pathway upon continuous stimulation by tetrapyrroles (von Gromoff et al.,
2008).
Additionally, two yellow mutants (chlM-1, chlM-2) defective in CHLM encoding
Mg-protoporphyrin IX methyltransferase (MgPMT) were identified (Meinecke et al., 2010).
The mutants do not accumulate chlorophyll, are yellow in the dark and dim light, and their
5 1. Summary 6
growth is inhibited at higher light intensities. They accumulate MgProto, the substrate of
MgPMT, and this may be the cause for their light sensitivity. In the dark, both mutants
showed a drastic reduction in the amounts of core proteins of photosystem I (PSI) and
photosystem II (PSII) and light-harvesting chlorophyll a/b-binding proteins. However, LHC
mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC
and other genes that were expressed at higher levels in the mutants during dark incubation
was attenuated in the initial phase of light exposure. No regulatory effects of the
constitutively 7- to 18-fold increased MgProto levels on gene expression were detected,
supporting previous results in which MgProto and heme in Chlamydomonas were assigned
roles as second messengers only in the transient activation of genes by light. The
constitutively enhanced MgProto levels do not result in an increased induction of genes after
a dark-to-light shift. The genes are already maximally induced by light, so enhanced
tetrapyrrole pool levels have no additional effect on gene expression (Meinecke et al., 2010).
In the other 34 mutants it was shown that continuous alterations of steady state levels of
tetrapyrroles did not result in a deregulation of gene expression.
To explore the question how important the regulation of genes by MgProto and heme is, we
measured the impact of MgProto and hemin feeding in the dark on changes in gene
expression at the genomic level using custom-made microarray. About 8% of the 10.000
genes represented on the microarray showed a transient up- or down-regulation with a fold
change equal to or above four (p  0.05). The two most prominent groups of regulated genes
were those where both MgProto and hemin caused either up- or down-regulation. Another
group consists of genes down-regulated by MgProto and a fourth group are genes
up-regulated by hemin. 415 of the 799 responding genes were also regulated by heat shock
and 91% of those in the same direction. These data suggest a role of MgProto and heme in the
quantitative changes of the proteome. Most prominent among the functional groups of
tetrapyrrole-regulated genes are those involved in protein folding and protein degradation.
Striking is the virtual absence of regulated genes that encode constituents of the
photosynthetic apparatus. This and the transient nature of changes in gene expression
observed upon feeding of the tetrapyrroles suggest a signaling role of these plastid
compounds in the adaptation of the alga to alterations in the environment.


6 2. Introduction 7
2 Introduction
2.1 Tetrapyrrole biosynthesis in photosynthetic organisms
Tetrapyrroles are common to all living organisms. They are responsible, inter alia, for oxygen
transport (heme), electron transport (cytochrome c) and, most fundamentaly, for
photosynthesis (chlorophyll). Photosynthetic organisms synthesize (bacterio-) chlorophyll,
heme, and phycobilins, the major tetrapyrroles in nature (von Wettstein D., 1995; Grimm,
1998). Indeed, life as we know it, would not exist on this planet without chlorophylls and
bilins (e.g., phycocyanin, which acts as a light harvesting pigment in algae). Chlorophyll is
the most abundant tetrapyrrole on earth. The pigment chlorophyll is responsible for light
absorption and energy transduction during photosynthesis. The porphyrins heme and
sirohaem are cofactors for proteins in diverse cellular processes including respiration
(cytochromes), cellular detoxification (e.g., catalase, peroxidase) and assimilation of
inorganic nitrogen and sulfur from the environment (nitrite and sulfite reductases).
Phytochromobilin belongs to the third class of plant tetrapyrroles, the phycobilins. In contrast
to chlorophylls and hemes, phycobilins are linear pigments. Phytochromobilin is the
chromophore of the phytochrome family of red/far-red photoreceptors (Terry et al., 1993).
The tetrapyrrole biosynthetic pathway leads to the synthesis of a number of important
products including chlorophylls and hemes (Fig. 1). The enzymatic steps and the genetic basis
of tetrapyrrole biosynthesis are well characterized (Suzuki et al., 1997; Skinner and Timko,
1998; Herman et al., 1999; Eckhardt et al., 2004; Lohr et al., 2005). Genes for virtually all of
the enzymes have been identified in higher plants. All enzymes involved are encoded by the
nucleus. The pathway is tightly regulated to ensure a continuous cofactor supply to the
cognate apoproteins whilst avoiding the phototoxic accumulation of intermediates (Cornah et
al., 2003; Tanaka and Tanaka, 2007). The tetrapyrrole biosynthetic pathway consists of four
different parts: (1) formation of 5-aminolevulinic acid (ALA), which is the committed step for
the synthesis of all tetrapyrroles, (2) formation of protoporphyrin IX (Proto), (3) formation of
chlorophyll and (4) formation of heme (Fig. 1). The three major tetrapyrrole end products,
chlorophyll, heme and phycobilins are synthesized in a branched metabolic pathway with
Proto as the last common precursor (Papenbrock and Grimm, 2001; Vavilin and Vermaas,
2002; Grossman et al., 2004; Beale, 2005).


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