Thesis
Reference
Regulation of chlorophyll synthesis in Chlamydomonas reinhardtii: the role of FLP and GUN4
CEOL, Mauro
Abstract
Le sujet de cette thèse est la régulation de la synthèse de la chlorophylle dans l'algue unicellulaire Chlamydomonas reinhardtii. En particulier, je me suis concentré sur les protéines GUN4 et FLP. GUN4 stimule l'activité de la MG-chelatase, la première enzyme impliquée dans la synthèse de la chlorophylle. FLP est un régulateur négatif de la synthèse de la chlorophylle et l'expression de cette protéine est régulée par un signal chloroplastique dépendant de l'accumulation des précurseurs de la chlorophylle. Nous avons montré que l'absence de GUN4 limite l'accumulation de la chlorophylle dans la cellule et qu'un niveau réduit de FLP provoque l'accumulation de niveaux élevés de différents précurseurs de la chlorophylle. Nous avons également identifié deux régulateurs putatifs de l'épissage alternatif de FLP.
CEOL, Mauro. Regulation of chlorophyll synthesis in Chlamydomonas reinhardtii: the role of FLP and GUN4. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4158
URN : urn:nbn:ch:unige-50772
DOI : 10.13097/archive-ouverte/unige:5077
Available at:
http://archive-ouverte.unige.ch/unige:5077
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITE DE GENEVE
Département de Biologie Moléculair
eFA CULTE DES SCIENCES
Professeur Jean-David Rochaix
Regulation of chlorop hyll synthesis in Chlamydomonas reinhardtii : the role of FLP and GUN4
TH ESE
Présentée à la Faculté des sciences de l’U niversité de G enève Pour obtenir le grade de Docteur ès Sciences, mention biologie
Par M auro Ceol
de l’Italie
Thèse n°
4158
G enève
A telier d'impression ReproMail
2009
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ENGLISH SUMMARY... 5
RESUMÉ DE LA THÈSE ... 7
I . GENERAL INTRODUCTION ... 9
CHLAMYDOMONAS REINHARDTII, A MODEL ORGANISM FOR PHOTOSYNTHESIS... 10
THE CHLOROPLAST... 13
OXYGENIC PHOTOSYNTHESIS... 15
The light reactions... 16
The dark reactions... 17
CHLOROPHYLL SYNTHESIS... 18
Regulation of the chlorophyll synthesis... 21
Chlorophyll synthesis in Chlamydomonas ... 24
RETROGRADE SIGNALING... 25
Overview of the known chloroplast-to-nucleus signals... 27
Plastid protein synthesis dependent signals...27
Reactive oxygen species dependent signaling...29
Signaling dependent on the redox state of the photosynthetic electron transport chain...30
Tetrapyrrole biosynthetic pathway dependent retrograde signaling...31
I I. ARTICLES AND RESULTS ... 37
ARTICLE 1:THE FLP PROTEINS ACT AS REGULATORS OF CHLOROPHYLL SYNTHESIS IN RESPONSE TO LIGHT AND PLASTID SIGNALS IN CHLAMYDOMONAS... 38
SEARCH FOR PROTEINS INVOLVED IN FLP REGULATION... 55
Genetic approach... 55
Biochemical approach: Experimental design... 55
Enrichment for nucleic acid binding proteins from total soluble extract...57
GRNA Chromatography ...59
In silico analysis of the candidates ...61
In-vitro binding assay of NLP ...65
MATERIALS AND METHODS... 68
ARTICLE 2:GUN4 IS REQUIRED FOR EFFICIENT CHLOROPHYLL SYNTHESIS AND PHOTOAUTOTROPHIC GROWTH IN CHLAMYDOMONAS REINHARDTII... 73
III . GENERAL DISCUSSION... 123
THE FLP PROTEINS REGULATE CHLOROPHYLL SYNTHESIS IN CHLAMYDOMONAS REINHARDTII... 124
NLP AND RORNP-3: TWO CANDIDATES FOR THE REGULATION OF FLP ALTERNATIVE SPLICING... 128
GUN4 ENSURES EFFICIENT CHLOROPHYLL SYNTHESIS AND PROPER BALANCING OF THE PHOTOSYNTHETIC ELECTRON TRANSPORT CHAIN... 130
REFERENCES ... 133
IV. APPENDIX ... 143
ACKNOWLEDGEMENTS ... 159
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English summary
During my thesis I studied the mechanisms that regulate chlorophyll synthesis. All organisms that perform oxygenic photosynthesis are facing an important challenge: how to produce chlorophyll without suffering from oxidative stress. Chlorophyll is a highly photodynamic molecule that is able to transfer an electron to an oxygen molecule, thus creating reactive oxygen species that can severely damage the cell. To reduce the probability of this event the cell should produce coordinated amounts of chlorophyll and chlorophyll-binding proteins. This is already challenging in photosynthetic bacteria, but is even more complicated in eukaryotic organisms, in which the major part of the chloroplastic proteins is encoded by the nuclear genome. These organisms evolved signaling mechanisms to coordinate gene expression between the nucleus and the chloroplast. The nucleus controls chloroplast gene expression mainly by supplying the factors required for its transcription, splicing and translation. Moreover the proteins involved in the assembly of the chloroplast complexes are often nucleus-encoded.
Interestingly, the chloroplast modulates nuclear gene expression by sending signals to the nucleus. The causes of these signals have been identified within the chloroplast, but the nature of the signals is still elusive.
My work has been focused on two chlorophyll synthesis regulators: FLP and GUN4.
The Flp gene encodes two proteins that are produced by alternative splicing of a short exon of 36 nucleotides. These proteins are the orthologues of Flu of Arabidopsis. They negatively regulate the first step of chlorophyll synthesis in response to the accumulation of protochlorophyllide. Thus they prevent the accumulation of high concentrations of chlorophyll photoreactive precursors. We have shown that the expression and the alternative splicing of this gene is regulated by light and by tetrapyrrole-dependent retrograde signals. Indeed in mutants affected in chlorophyll synthesis the amount of the two FLP isoforms varies depending on the presence of specific molecules. We have correlated their expression with the accumulation of
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precursors of the tetrapyrrole pathway that are common with heme synthesis and the alternative splicing with the presence of chlorophyll-specific precursors.
GUN4 is a protein that stimulates the activity of the Mg-chelatase, the first enzyme committed specifically to chlorophyll synthesis. It does so by binding the substrate (protoporphyrin IX) and the product (Mg-protoporphyrin IX) of the chelation reaction and by facilitating the release of the Mg-protoporphyrin IX. In Arabidopsis this mutation causes a drastic decrease in chlorophyll content, loss of coordinated gene expression between the chloroplast and the nucleus and severely affects photoautotrophic growth. We have shown that in Chlamydomonas the absence of GUN4 has a relatively mild effect on the chlorophyll content, but photoautotrophic growth is strongly affected. This phenotype is most probably not due to photodamage, as in dim light on acetate-containing medium the gun4 mutant grows like the wild type. We have shown that in the mutant the photosynthetic activity is strongly affected, an observation which can explain the impaired photoautotrophic growth phenotype.
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Resumé de la thèse
Durant ma thèse j’ai étudié les mécanismes qui régulent la synthèse de la chlorophylle. Tous les organismes accomplissant la photosynthèse oxygénique font face à un important problème:
comment produire de la chlorophylle sans souffrir du stress oxydatif. La chlorophylle libre est une molécule fortement photodynamique qui est capable de transférer un électron à une molécule d’oxygène, créant ainsi des dérivés réactifs de l’oxygène (ROS) pouvant sévèrement endommager la cellule. Afin d’éviter ces dommages, la cellule se doit de produire des quantités équivalentes de chlorophylle et de protéines liant la chlorophylle. Ce problème est d’autant plus compliqué dans une cellule eucaryote, par rapport aux cellules procaryotes, de part le fait que la majeure partie des protéines chloroplastiques est codée par le génome nucléaire. Ces organismes ont donc développé des voies de signalisation afin de coordonner l’expression des gènes dans le noyau et le chloroplaste. Le noyau contrôle l’expression génique dans le chloroplaste principalement en fournissant des facteurs nécessaires à la transcription, à l’épissage et à la traduction. De plus, les protéines impliquées dans l’assemblage des complexes chloroplastiques sont souvent codées par le noyau. L’expression génique nucléaire est par ailleurs modulée par des signaux chloroplastiques (signaux rétrogrades), qui bien qu’identifiés dans le chloroplaste, restent globalement inconnus.
Mon travail a porté sur deux régulateurs de la synthèse de la chlorophylle : FLP et GUN4.
Le gène Flp qui code pour deux protéines produites par épissage alternatif d’un court exon de 36 nucléotides, est l’orthologue de Flu chez Arabidopsis. Ces deux protéines répriment la première étape de la synthèse de chlorophylle en réponse à l’accumulation de protochlorophyllide, empêchant ainsi l’accumulation à haute concentration de précurseurs photoréactifs de la chlorophylle. Nous avons montré que non seulement l’expression mais aussi l’épissage alternatif de ce gène sont régulés par la lumière et par des signaux rétrogrades
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dépendant des tétrapyrroles. En fait, dans des mutants affectés dans la synthèse de chlorophylle, la quantité des deux isoformes FLP varie en fonction de la présence d’intermédiaires de porphyrines spécifiques. Nous avons montré que l’ expression de ces deux isoformes est corrélée à l’accumulation de précurseurs de la voie de synthèse des tétrapyrroles qui sont communs à la synthèse de l’hème, tandis que l’épissage alternatif est corrélé à la présence de précurseurs spécifiques à la synthèse de chlorophylle.
La protéine GUN4 stimule l’activité de la Mg-chélatase, la première enzyme spécifiquement impliquée dans la synthèse de la chlorophylle, en liant le substrat (protoporphyrine IX) et le produit (Mg-protoporphyrine IX) de la réaction de chélation et en facilitant la relâchement du produit. Chez Arabidopsis, cette mutation produit une réduction drastique de la quantité de chlorophylle, la perte de l’expression génique coordonnée entre le noyau et le chloroplaste et affecte sévèrement la croissance photoautotrophique. Nous avons montré que chez Chlamydomonas, l’absence de GUN4 a un effet relativement modéré sur la quantité de chlorophylle mais la croissance photoautotropique est fortement affectée. Ce phénotype n’est probablement pas dû aux photodommages car, à une lumière de faible intensité sur un milieu contenant de l’acétate, le mutant gun4 pousse comme le type sauvage. Nous avons constaté que, pour le mutant, l’activité photosynthétique est fortement affectée. Cette observation peut expliquer le phénotype de croissance photoautotrophique.
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I . General introduction
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Chlamydomonas reinhardtii, a model organism for photosynthesis
Chlamydomonas reinhardtii is a unicellular green alga with a diameter of ~10 m, it can be found in fresh-water and soil. A Chlamydomonas cell contains a single chloroplast, which takes up half of the total cellular volume, several mitochondria and a nucleus, it is surrounded by a proteinaceus cell wall and it possesses two flagella that are used by Chlamydomonas for motility and mating.
Figure 1: A. Schematic representations of a Chlamydomonas reinhardtii cell, the main cellular components are indicated by arrows (adapted from http://www.metamicrobe.com). B.
Microscopy picture of a cell of Chlamydomonas reinhardtii (adapted from http://www.biol.s.u- tokyo.ac.jp).
Chlamydomonas has been extensively used as a model for studying the regulation of chloroplast gene expression and the biogenesis of the photosynthetic apparatus. For the study of photosynthesis the most important advantage of working with Chlamydomonas is its ability to grow under non-photoautotrophic conditions. In fact in this organism photosynthesis is not
11 essential if it is supplied with a reduced carbon source, for example acetate in the common growth media, TAP (Tris-Acetate-Phosphate). Another advantage of Chlamydomonas is that it can produce chlorophyll in the dark, as it possesses a light-independent protochlorophyllide oxido-reductase (LIPOR), and can develop a functional chloroplast also in the absence of light.
In contrast higher plants do not possess this enzyme and develop etioplasts instead of chloroplasts in the dark. The presence of chlorophyll enables Chlamydomonas to assemble a functional photosynthetic apparatus in the dark, a remarkable feature for the study of photosynthesis, as it allows for the growth of photosynthetic mutants that would die in the presence of light due to photodamage, as for example any mutant affected at the level of photosystem I function.
Figure 2: schematic representation of the life cycle of Chlamydomonas reinhardtii: on the right is depicted the vegetative stage (asexual reproduction) and on the left the reproductive stage (adapted from http://io.uwinnipeg.ca).
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Another advantage of using Chlamydomonas reinhardtii is its genetics. In fact Chlamydomonas cells are haploid for the major part of their life cycle. In this way mutations are readily uncovered without the requirement of crosses to obtain homozygous mutants. Chlamydomonas can be forced to mate by nitrogen starvation. Under these conditions the cells differentiate into gametes of two different mating types (plus and minus) that can mate to produce a diploid zygote. The zygote generates four spores by meiosis. These spores inherit the nuclear genes in a Mendelian way whereas the chloroplast and mitochondrial genomes are mostly uniparentally inherited from the mating type plus and minus parent, respectively. A powerful genetic tool comes also from the sequencing data as all the three genomes of Chlamydomonas are fully sequenced (Merchant et al., 2007). The nuclear genome has a size of 121 megabases and slightly less than 9000 genes are predicted. The GC content of this DNA is 64% (Merchant et al., 2007). The chloroplastic genome with its 203 kilobases is larger than many plant plastid genomes and it is present in ~80 copies per chloroplast. It is AT-rich (65.4%) and contains 99 genes (Maul et al., 2002).
Of great importance is the availability of techniques for the transformation of the three genomes of Chlamydomonas. Nuclear transformation occurs through random insertion of the transforming DNA. For this reason it has been used to create mutants that are tagged by the insertion. On the other hand chloroplastic transformation occurs through homologous recombination making it possible to characterize non-essential chloroplastic genes. These techniques allow the complementation of mutations in any of the three cellular organelles. The use of genetic and DNA transformation technologies allows for the combination of different nuclear and plastid mutations that are impossible to obtain in higher plants. Finally Chlamydomonas is well suited for biochemical studies as it can be grown in large quantities in liquid culture.
13 All these features make Chlamydomonas reinhardtii a powerful model organism for the study of plastid biogenesis, in particular of the entire process of photosynthesis and its regulation:
from the control of nuclear and chloroplastic genes, to the assembly of the photosynthetic complexes, to the energetic transition between different electron carriers in the electron transport chain (Hippler et al., 1998; Rochaix, 2002).
The Chloroplast
In photosynthetic eukaryotes photosynthesis takes place in the chloroplast, an organelle that is present in all green tissues of these organisms. The chloroplast is compartmentalized in a series of specialized domains by a complex network of membranes.
The organelle is surrounded by a double membrane system called the envelope; the outer envelope is highly permeable and allows for free diffusion of molecules between the cytoplasm and the inter-membrane compartment. The inner envelope physically separates the internal part of the chloroplast from the cytoplasm and it contains specific transporters to regulate the exchange of metabolites between the two compartments. Two important events at the level of the envelope are the import of proteins in the chloroplast, since the chloroplastic genome encodes only a minor part of the proteins necessary for chloroplast functions, and the export of triose-phosphates from the chloroplast to the cytoplasm, where they are converted to sucrose and eventually exported to other plant tissues.
The aqueous space inside the chloroplast is the stroma in which the chloroplast genome is contained and where chloroplast protein synthesis and the reactions of the dark phase of photosynthesis occur.
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Figure 3: representation of the chloroplast and its internal structures: it is possible to note the inner and outer envelope and the thylakoids network. On the right an electron microscopic image of a chloroplast (from http://www.wellesley.edu)
Separated from the stroma by the membranes of the thylakoids is the lumen. In this compartment oxygen is produced from water by the oxygen evolving complex associated with photosystem II. The thylakoids are organized in grana and lamellae: the grana are composed of tightly packed membranes, whereas the lamellae are single membranes that connect the grana.
The photosynthetic complexes are all embedded in the thylakoid membranes and the electrons are transported from the lumen to the stroma during photosynthesis.
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Oxygenic photosynthesis
Photosynthesis comprises a complex sequence of reactions that allows plants, algae and photosynthetic bacteria to grow using sun light as an energy source. These organisms are called photoautotrophs and they use photosynthesis to convert the light radiation into chemical energy, which can then be used to produce the organic molecules they need for growth.
Oxygenic photosynthesis consists of a series of reactions of oxido-reduction that use water molecules as a primary source of electrons. The electrons are transferred through a series of carriers to an acceptor molecule that is then used to reduce carbon dioxide in order to produce carbohydrates. The whole oxygenic photosynthesis can be summarized by the formula:
6H2O + 6CO2 C6H12O6 + 6O2
A by-product of the reaction is molecular oxygen (O2), the key element for the life of many organisms on earth.
The photosynthetic process can be schematically divided in two phases: the light reactions and the dark reactions. The light reactions require light as an energy source to extract electrons from water molecules that can be used to reduce NADP+ to NADPH. At the same time the photosynthetic electron flow generates an electro-chemical gradient across the thylakoid membrane by pumping protons into the lumen. This gradient is used by the ATP-synthase to generate ATP from ADP and inorganic phosphate.
The dark reactions are in principle light-independent and use the NADPH and the ATP generated by the light reaction to produce glyceraldehyde-3-phosphate from CO2 in a process that is called the Calvin-Benson cycle (Benson and Calvin, 1950). However the enzymes of this cycle need to be activated by light.
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The light reactions
The light reactions can be seen as a charge separation across the thylakoid membrane between the negatively charged electrons and the positively charged protons extracted from the water molecules.
The electron transport occurs via three multiprotein complexes embedded in the thylakoid membrane: photosystem II (PSII), the cytochrome b6f complex (cyt b6f) and photosystem I (PSI). The electrons are extracted from water molecules at the level of the manganese cluster of the Oxygen-Evolving Complex through the high oxidizing power of the reaction center of PSII.
The reaction center is the converging point for the energy harvested by the antennae of the photosystem and when oxidized possesses a redox potential high enough to oxidize a H2O molecule. The electrons extracted can be excited to a high energetic level thus allowing them to be transferred through PSII to the plastoquinone pool. The reduced plastoquinone can diffuse in the membrane and reach the cyt b6f complex, which transfers the electrons subsequently to plastocyanin, a lumenal protein that can then move to photosystem I. Once it is bound to photosystem I at the docking site, plastocyanin can be oxidized by the reaction center of PSI and the electrons are pumped through the membrane by a similar mechanism occurring in PSII and transferred to ferredoxin. The reduced ferredoxin is then used by the ferredoxin-NADP+ oxido-reductase to produce NADPH.
In this series of events there are two excitation steps, one at the level of PSII and one at the level of PSI, the whole electron transport chain can be visualized on a redox potential diagram called the Z-scheme (Hill and Bendall, 1960).
The transport of electrons across the membrane is coupled to an influx of protons in the thylakoid lumen. The protons accumulated create an electro-chemical gradient between the
17 lumenal and the stromal compartments. The protons can move to the stromal side through the channel created by the ATP-synthase, and this flux is used by the enzyme to produce ATP.
Figure 4: the Z-scheme (from http://www.uqtr.ca/labcarpentier), the electron extracted from water is transferred to the reaction center of the PSII (P680). A photon excites P680 and the electron is transferred from one acceptor to the other to reach the reaction center of PSI (P700). At this stage a second photon excites P700 with concomitant charge separation and electron transfer to ferredoxin and NADP+.
The dark reactions
The Calvin-Benson cycle takes place in the stroma of the chloroplast. The enzymes that catalyze the reactions use the ATP and the NADPH generated by the light reactions to produce reduced carbon compounds in the form of glyceraldehayde-3-phosphate starting from CO2 and water.
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The key enzyme of the cycle is the ribulose-1,5-bisphosphate carboxylase/oxygenase, or RuBisCO, which catalyzes the addition of a carbon group derived from the CO2 to the pentose ribulose-1,5 bisphosphate to give rise to two triose-phosphates, 3-phosphoglycerate
Figure 5: scheme of the Calvin-Benson cycle, the red balloons represent carbon atoms (adapted from http//www.uic.edu)
Chlorophyll synthesis
Chlorophyll is the most abundant pigment in plant leaves and is responsible for the capture of the light energy and for its transfer to the reaction center of the photosystems. The reaction centers themselves are composed of two chlorophyll molecules that are arranged with the tetrapyrrolic rings parallel to each other, in this way they act like a single excitable molecule
19 able to promote electrons to high energetic states, thus allowing their transfer to other electron carriers in the electron transport chain.
Chlorophyll is produced through the tetrapyrrole pathway, which can generate four different tetrapyrroles: siroheme, heme, phytochromobilin and chlorophyll. Siroheme is the cofactor of nitrite and sulfite reductase, heme is an electron carrier involved in the electron transport chain of photosynthesis and in the respiratory chain, phytochromobilin is a linear tetrapyrrole and is the cofactor of the phytochrome family of photoreceptors.
In figure 6 the tetrapyrrole pathway of higher plants is depicted. It is important to note that the precursor of all tetrapyrroles is glutamyl tRNA, which is also involved in protein synthesis in the chloroplast. This molecule connects the production of the chlorophylls with the translation of some of the apoproteins that bind it.
The first enzyme of the pathway is glutamyl tRNA reductase (GluTR), whose product is used by the glutamate 1-semialdehyde aminotransferase (GSA-AT) to produce 5-aminolevulinic acid (ALA).
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Figure 6: overview of the tetrapyrrole pathway of higher plants. Some precursors and enzymes have been omitted (adapted from http://biochemsoctrans.org). The monopyrrole porphobilinogen is produced from the glutamyl tRNA, this molecule is then use to synthesize the cyclic tetrapyrrole protoporphyrin IX. From protoporphyrin IX the activities of the Mg- chelatase and of the Fe-chelatase lead to the production of chlorophyll and heme respectively, in separate branches. Early in the pathway a branching point leads to the production of the cyclic tetrapyrrole siroheme, the cofactor of nitrite and sulfite reductases.
Two molecules of ALA are condensed to obtain porphobilinogen, a monopyrrole. Four molecules of porphobilinogen are linked together to produce the first cyclic tetrapyrrole,
21 uroporphyrinogen III. From this molecule the first branching in the pathway occurs in which two enzymatic steps lead to the generation of siroheme.
In a few steps the uroporphyrinogen III is converted to protoporphyrin IX, the last common precursor between chlorophyll and heme. The Mg branch of the pathway continues with the insertion of a Mg2+ in the tetrapyrrolic ring of protoporphyrin IX by the Mg-chelatase (MgCh).
The Mg-protoporphyrin IX obtained is methylated by the Mg-protoporphyrin IX methyltransferase (MgMT) and then converted to protochlorophyllide a by the Mg- protoporphyrin IX monomethyl ester cyclase (MgCy). The subsequent reduction of the protochlorophyllide to chlorophyllide is light dependent in angiosperms. Once light triggers the activation of the protochlorophyllide reductase (POR), chlorophyllide a is produced. At this step two different enzymes can use chlorophyllide a as substrate: chlorophyll synthase can add a phytol chain to chlorophyllide a to obtain chlorophyll a and the chlorophyllide a oxygenase (CAO) can oxidize the chlorophyllide a to produce chlorophyllide b, which can be used by the chlorophyll synthase to generate chlorophyll b.
The heme branch of the pathway begins with the Fe-chelatase (FeCh), which catalyzes the insertion of a Fe2+ in the tetrapyrrolic ring of protoporphyrin IX to produce heme. The cyclic molecule of heme can be opened to generate phytochromobilin.
Regulation of the chlorophyll synthesis
The tetrapyrrole pathway is tightly regulated because the requirement of specific tetrapyrroles can vary depending on the cell types or on the environmental cues and because many intermediates and the final products can be excited by light and this may lead to the formation of toxic radicals and singlet oxygen (Masuda and Fujita, 2008).
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One of the most important points of regulation is the formation of ALA, controlling the very first step of the pathway. This presents an important advantage because almost all the intermediates can generate reactive radicals upon illumination. Thus, blocking the pathway at the beginning is an effective way of preventing photo-oxidative damage. For the synthesis of ALA the enzyme whose activity is regulated is GluTR. This is the first enzyme committed only to the tetrapyrrole pathway, given the fact that the conjugation of glutamate to tRNAGlu is shared with protein synthesis. GluTR is subjected to feedback regulation by the two branches of the tetrapyrrole pathway that lead to chlorophyll and heme. The fluorescence (flu) mutant of Arabidopsis accumulates protochlorophyllide in the dark. The protein encoded by the Flu gene is a small membrane-associated chloroplast protein that can interact directly with the GluTR based on the yeast two-hybrid system (Meskauskiene and Apel, 2002; Meskauskiene et al., 2001). The Flu protein acts in response to accumulation of protochlorophyllide by downregulating the activity of the GluTR. Interestingly a suppressor of flu was identified in a genetic screen (Wagner et al., 2004). This suppressor still accumulates protochlorophyllide in the dark, but to a much lower extent compared to the original flu mutant. The gene was shown to be allelic to the Hy1 locus, which is known to encode heme oxygenase, the first enzyme of the pathway that leads from heme to phytochromobilin (Goslings et al., 2004). The disruption of the heme oxygenase induces the accumulation of heme, and the increase in heme concentration inhibits the activity of GluTR independently from the Flu protein. This observation is in agreement with previous studies on GluTR where it was shown that heme can inhibit the enzyme activity in vitro (Pontoppidan and Kannangara, 1994; Vothknecht et al., 1998).
Another important regulatory step is the branching point between chlorophyll and heme. The activity of MgCh in fact is controlled by several chloroplastic cues. MgCh is composed of three subunits CHLH, CHLI and CHLD, with the catalytic site located in the ChlH subunit. In addition, the Gun4 protein is also part of this complex and is an important enhancer of the
23 activity of the enzyme. The activity of the MgCh is controlled by the redox state of the chloroplast, which is an indicator of the efficiency of photosynthesis. The ATPase activity of the CHLI subunit was shown to be redox-dependent in vitro, being active in a reducing environment and largely inactive in an oxidizing one. Moreover, in the same studies the MgCh activity was stimulated in isolated chloroplasts upon reduction (Ikegami et al., 2007; Kobayashi et al., 2008). Other important effectors that act on the MgCh are the concentration of free Mg2+
and the ATP/ADP ratio. It was shown that the MgCh activity depends on the concentration of stromal Mg2+ (Reid and Hunter, 2004). Interestingly, the Mg2+ concentration required for a full activation of the enzyme is significantly lowered by the presence of the Gun4 protein in the complex (Davison et al., 2005; Verdecia et al., 2005). Another effect of the concentration of Mg2+ is on the localization of the MgCh. In fact the CHLH subunit has been reported to be membrane associated at a concentration of 5 mM Mg2+ and to be localized in the stroma at a concentration of 1 mM (Gibson et al., 1996; Nakayama et al., 1998). It is possible that the association of MgCh to the membrane is required for the efficient transfer of protoporphyrin IX from the membrane-associated protoporphyrinogen IX oxidase to MgCh (Tanaka and Tanaka, 2007). ATP is necessary to activate the MgCh (Reid and Hunter, 2004) and has a negative effect on the FeCh (Cornah et al., 2002). Upon illumination the concentration of free Mg2+ in the stroma increases (Ishijima et al., 2003) as well as the ATP/ADP ratio (Usuda, 1988). It is therefore likely that the concurrent increase in Mg2+ concentration and in the ATP/ADP ratio after a dark-to-light shift promotes the production of chlorophyll at the expense of heme in illuminated chloroplasts.
The regulation of chlorophyll synthesis appears to be achieved mainly by fine tuning the enzymatic activity of key steps in the pathway. Nevertheless a transcriptional coordination was observed for genes of enzymes involved in the pathway, in particular a tight correlation has been found between the genes coding for the glutamyl-tRNA reductase (HEMA1), for the CHLH subunit of Mg-chelatase (CHLH), for the chlorophyll a oxygenase (CAO), for GUN4
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and for the geranylgeranyl reductase (CHLP), the enzyme responsible of the production of the phytol pyrophosphate, a molecule essential for the synthesis of chlorophylls, carotenoids and tocopherols (Matsumoto et al., 2004). The relevance of this coordination is still only partially understood, but the fact that also some chlorophyll binding proteins are co-expressed with these genes suggest that this is another important level of regulation for chlorophyll biosynthesis (Masuda and Fujita, 2008).
Chlorophyll synthesis in Chlamydomonas
The pathway for the synthesis of chlorophyll in Chlamydomonas differs from the one of higher plants mainly because this organism is able to produce chlorophyll also in the dark. This is possible thanks to the presence of a light-independent protochlorophyllide reductase (LI-POR).
This enzyme is present in cyanobacteria, algae, ferns and gymnosperms, but is absent in angiosperms. It is composed of three subunits, CHLN, CHLB and CHLL, which are encoded in the genome of the chloroplast in Chlamydomonas (Choquet et al., 1992; Li et al., 1993; Suzuki and Bauer, 1992). In addition to the three subunits of the enzyme, seven nuclear loci are essential for the synthesis of chlorophyll in the dark in Chlamydomonas, Y1 and Y5-Y10 (Cahoon and Timko, 2000). Mutations in these genes, with the exception of Y7, are characterized by the absence of CHLL. It is possible that the Y genes encode factors that are essential for the production or the assembly of the CHLL subunit of the LI-POR.
Chlorophyll biosynthesis has been extensively studied in Chlamydomonas thanks to the possibility of growing chlorophyll-deficient mutants in the laboratory. In Table 1 some of the mutants of the Mg branch of the tetrapyrrole pathway are summarized.
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Mutant(s) Gene affected Phenotype Reference
brs1, chl1 Mg-chelatase subunit H
Accumulation of high amount of Protoporphyrin IX, dies in the light
(Chekounova et al., 2001; Wang et al.,
1974) brc1 Not known
Accumulation of Protoporphyrin IX and of Mg-Protoporphyrin IX, light sensitive, still able to produce
chlorophyll in the light
(Wang et al., 1974)
chlL LI-POR
subunit L
Accumulate protochlorophyllide in the dark, yellow-in-the-dark, green
in the light
(Suzuki and Bauer, 1992)
chlB LI-POR
subunit B
Accumulate protochlorophyllide in the dark, yellow-in-the-dark, green
in the light
(Li et al., 1993)
y1 Not known
Accumulate protochlorophyllide in the dark, yellow-in-the-dark, green
in the light
(Cahoon and Timko, 2000)
pc1 POR (Li and Timko, 1996)
Table 1. List of some of the mutants of Chlamydomonas affected in the Mg branch of the tetrapyrrole pathway. The genes affected are indicated if known and the phenotypes are summarized.
A difference compared to angiosperms that is important for this thesis is the fact that Chlamydomonas has two FLP proteins, and these proteins arise from alternative splicing of a precursor transcript from a single gene (Falciatore et al., 2005).
Retrograde signaling
The endosymbiotic theory describes the chloroplast as the result of the uptake of an ancient photosynthetic prokaryote by an eukaryotic unicellular organism. Instead of being consumed as
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nutriment, the ancient bacteria established a symbiotic relationship with the eukaryotic host.
The symbiosis became stable and in time the prokaryote became what is now known as the chloroplast. The original photosynthetic prokaryote presumably contained all the genes necessary for an independent photoautotrophic life. During time most of these genes have been lost or transferred to the host eukaryotic nucleus, leading to a complete dependency on the host for the chloroplast metabolism. Nowadays the chloroplast genomes contain 100-150 open reading frames, mainly coding for photosynthetic proteins or for factors necessary for chloroplast gene expression. On the other hand the chloroplast contains more than 3000 polypeptides encoded by the nucleus, translated in the cytoplasm and transported across the envelope to the stroma or the thylakoids (Abdallah et al., 2000; Martin et al., 2002). The presence of proteins of different origin in the chloroplast represents a serious challenge for the photosynthetic organism: indeed chloroplast complexes involved in metabolic pathways and in photosynthesis are composed of subunits encoded both by nuclear and chloroplast genes. The synthesis of proteins that work together should be coordinated between the nucleus and the chloroplast and this coordination can be achieved only with the exchange of signals between the two cellular compartments.
These signals can be anterograde, from the nucleus to the chloroplast, or retrograde, from the chloroplast to the nucleus. Anterograde signals include mainly factors necessary for chloroplast gene expression as the nucleus encodes most of the components of the transcription and translation machineries as well as many factors that are required for the expression of specific chloroplast genes (Goldschmidt-Clermont, 1998; Leon et al., 1998). The nature of the retrograde signal is more elusive. Today it is clear that several different signals arise from the chloroplast to modulate the expression of nuclear genes encoding chloroplast proteins.
Processes leading to these signals have been identified, but despite the efforts of many laboratories worldwide it is still unknown how the signals exit the chloroplast to trigger a nuclear response.
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Overview of the known chloroplast-to-nucleus signals
Several chloroplast retrograde signals have been identified so far: one dependent on plastid protein synthesis, a second on hydrogen peroxide, a third on singlet oxygen, a fourth on the redox state of the electron transport chain and a fifth on the tetrapyrrole biosynthetic pathway.
Figure 7: Model of the retrograde signaling pathways discussed below.
Plastid protein synthesis dependent signals
Plastid protein synthesis was proposed to affect nuclear gene expression based on studies on the barley mutant albostrians. This mutant is deficient in chloroplast ribosomes and therefore
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impaired in chloroplast protein synthesis (Bradbeer et al., 1979). In the pigment-deficient leaves, the mutant seedlings fail to express several nucleus-encoded chloroplast proteins that are normally induced by light, suggesting the absence of a signal required for correct light- induced gene expression in the ribosome-deficient cells (Hess et al., 1994).
Other evidences for the existence of a plastid protein synthesis-dependent retrograde signal derive from the use of transcriptional or translational inhibitors specific for the plastid ribosomes. Treatment of barley seedlings with the plastid RNA polymerase inhibitor tagetitoxin prevents the induction of nuclear genes that are expressed during early phases of development (Rapp and Mullet, 1991). Inhibition of chloroplast translation by treatment with chloramphenicol or lincomycin or mutations that affect essential components of the plastid translational machinery also have inhibitory effects on the expression of nuclear genes encoding chloroplast proteins (Sullivan and Gray, 1999). It is interesting to note that these treatments have an effect on gene expression only if applied within 3 days after germination, suggesting the involvement of a factor that is only present in the early phases of seedling development (Beck, 2005; Pogson et al., 2008).
Recently the mutant genome uncoupled 1 (gun1) has been reported to release the inhibitory effect of translational inhibitors on nuclear gene expression. In the same studies the signal mediated by GUN1 was shown to have inhibitory effects on cryptochrome CRY1 signaling, thus implicating an interaction between plastid protein synthesis and the light signaling pathways (Ruckle et al., 2007; Ruckle and Larkin, 2009). GUN1 is a member of the pentatricopeptide-repeat (PPR) protein family, it is nucleus-encoded and imported in the chloroplast. In addition to the PPR domain, GUN1 contains a small Muts-related (SMR) domain that can bind strongly to DNA. The role of this binding is still unknown (Koussevitzky et al., 2007). Downstream effectors of GUN1 are still unknown. Some evidence suggests that the transcription factor ABI4 is implicated in this pathway, but it is unlikely that this is the only nucleo-cytosolic factor involved in GUN1 signaling (Koussevitzky et al., 2007).
29 Reactive oxygen species dependent signaling
The chloroplast is one of the major sources of reactive oxygen species (ROS) in plants.
Chlorophyll molecules excited to the triplet state can transfer their energy to oxygen (3O2) giving rise to singlet oxygen (1O2). This event occurs mainly at the level of photosystem II if the electron transport chain does not work efficiently (Krieger-Liszkay, 2005). Photosystem I can directly transfer an electron to oxygen instead of NADP+ creating superoxide ions (O2•-
), that are converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) (Apel and Hirt, 2004).
Because of its short half life, singlet oxygen is probably exclusively localized in the chloroplast, its role was difficult to separate from the role of other ROS due to the difficulties in generating it specifically in plant cells. Recently the use of the Arabidopsis mutant flu led to great advances in the understanding of the singlet oxygen signaling pathway. FLU is a negative regulator of chlorophyll biosynthesis (see previous chapters on the regulation of chlorophyll synthesis) and its absence leads to the accumulation of protochlorophyllide, which generates singlet oxygen upon illumination (Meskauskiene et al., 2001). Illuminated flu plants arrest their development and eventually die. This event was thought to be caused by irreversible photoxidative damage, but the discovery of suppressors of the flu phenotype highlights the existence of a singlet oxygen induced programmed cell death. The executer1/flu double mutant grows like the wild type in conditions that are inhibitory for the flu plants. EXECUTER1 (EX1) has been shown to be an integrator of singlet oxygen signaling from the chloroplast. In its absence the plant no longer arrests its growth in response to protochlorophyllide-dependent generation of singlet oxygen (Wagner et al., 2004). In Arabidopsis there is also a homologue of EX1, named EXECUTER2 (EX2). In a triple mutant flu/ex1/ex2 induction of almost all the nuclear genes responsive to singlet oxygen is abolished, strengthening the idea that EX1, and
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EX2, are essential components of the singlet oxygen dependent retrograde signaling pathway (Lee et al., 2007).
Hydrogen peroxide is produced by the photosynthetic apparatus under different stress conditions and changes in the expression of nucleus-encoded stress proteins or light-induced proteins have been linked to hydrogen peroxide (Slesak et al., 2007; Vandenabeele et al., 2003;
Vandenabeele et al., 2004). Studies with reporter genes have shown that application of exogenous hydrogen peroxide stimulates the expression of high light-inducible genes like ascorbate peroxidases APX1 and APX2, the zinc finger transcription factors ZAT10 and ZAT12 and the early light inducible protein ELIP2 (Davletova et al., 2005; Karpinski et al., 1999; Rossel et al., 2007). Recently an interaction between the singlet oxygen and the hydrogen peroxide signaling pathways has been proposed, in particular it has been shown with transcriptomic analysis that hydrogen peroxide has an antagonistic effect to singlet oxygen on nuclear gene expression (Laloi et al., 2007).
The interaction between retrograde signaling pathways increases the complexity of the chloroplast-to-nucleus communication and may explain the adaptability of the plant cell to many different environmental conditions.
Signaling dependent on the redox state of the photosynthetic electron transport chain
The redox state of the electron transport chain, and in particular of the plastoquinone pool is an indicator of the efficiency of photosynthesis. If the photosynthetic reactions are not working properly, modifications in the expression of nuclear genes may be necessary to adjust them. The photosynthetic electron transport chain was therefore a likely candidate as the origin of retrograde signals. Even if gene regulation linked to the redox state of the plastoquinone pool has been found, evidence for it seems to be largely dependent on the plant species, the experimental conditions and the developmental stage. Moreover the distinction between redox signals and other signaling pathways has proven very difficult (Pogson et al., 2008). In the
31 unicellular alga Dunaliella tertiolecta, it was shown that the expression of the LHCB gene is altered in response to reduction or oxidation of the plastoquinone pool (Durnford and Falkowski, 1997). In plants the regulation of some genes has been linked to the redox state of the plastoquinone pool. Indeed in Arabidopsis 54 genes have been shown to be strictly regulated by the redox state of the plastoquinone pool, whereas many more seemed to be regulated together with other chloroplast signals (Fey et al., 2005b). In Chlamydomonas the redox-dependent retrograde signal has been partially characterized using strains carrying mutations in the cytochrome b6f acceptor site Q0, or lacking the cytochrome b6f complex. These strains were unable to induce the genes of the tetrapyrrole biosynthetic pathway upon illumination (Shao et al., 2006). Interestingly this inhibitory effect is specific for the cytochrome b6f complex and independent from the redox state of the plastoquinone pool as treatment of the cells with DCMU (an inhibitor of electron transfer from PSII to the plastoquinone) or DBMIB (an inhibitor of electron transfer from plastoquinone to the cytochrome b6f) have no effect on the light induction of the genes analyzed (Shao et al., 2006).
Recently the STN7 kinase has been linked to long term adaptation and redox dependent retrograde signaling. This kinase is necessary for the state transition process, a short term adaptation to changing light conditions (Bellafiore et al., 2005). In the stn7 plants the regulation of a set of nuclear genes is perturbed and the plants show reduced growth if the plants are subjected repeatedly to light of different wavelengths. This has led to the hypothesis of a dual role for the STN7 kinase in short and long term adaptation (Bonardi et al., 2005; Rochaix, 2007; Wagner et al., 2008).
Tetrapyrrole biosynthetic pathway dependent retrograde signaling
The possible involvement of chlorophyll precursors in the regulation of nucleus-encoded chloroplast proteins was suggested by studies on dipyridyl- treated cells (dipyridyl inhibits the FeCh and the Mg-protoporphyrin IX monomethyl ester cyclase) and on protoporphyrin IX
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accumulating mutants of Chlamydomonas reinhardtii. In these conditions the light-dependent accumulation of the LHCB transcripts was prevented, this effect was correlated with the overaccumulation of cyclic tetrapyrroles, as the treatment with dyoxoheptanoic acid, an inhibitor of earlier steps in the tetrapyrrole pathway, had no effect on LHCB mRNA accumulation (Johanningmeier, 1988; Johanningmeier and Howell, 1984). Another proof of the role of chlorophyll precursors in the retrograde signaling in Chlamydomonas came from the studies on chloroplast HSP70. It was shown that the induction of this gene by light was impaired in the MgCh mutant brs1, but not in a mutant affected in an enzyme that acts in a subsequent step of the chlorophyll biosynthetic pathway. Moreover feeding of Mg- protoporphyrin IX and Mg-protoporphyrin IX methylester, but not of protoporphyrin IX in the dark was sufficient to induce the expression of HSP70 in the dark (Kropat et al., 1997, 2000). In the same studies a correlation was observed between the induction of the HSP70 gene and a transient increase in the cellular concentration of chlorophyll precursors upon illumination, supporting a model in which the accumulation of cyclic tetrapyrroles has an important role in triggering a signal to modulate nuclear gene expression (Kropat et al., 2000). Why protoporphyrin IX does not seem to have a role in retrograde signaling is more difficult to explain. In fact feeding the cells with this molecule causes an increase in the cellular level of Mg-protoporphyrin and Mg-protoporphyrin methyl ester, an event that, according to the model proposed, should lead to an induction of the transcription of the HSP70 gene which is however not observed. The explanation given by the authors is that feeding of Mg-protoporphyrin IX increases the cellular concentration of this tetrapyrrole in every compartment of the cell including the cytosol and the nucleus, whereas enzymatic conversion of protoporphyrin IX to Mg-protoporphyrin IX leads to an increase of the latter only in the chloroplast, and another factor, probably light, is required for the export of the Mg-protoporphyrin IX to the cytosol (Beck, 2005). Convincing evidence of the export of Mg-protoporphyrin IX from the chloroplast is still missing.
33 The characterization of the FLP proteins in Chlamydomonas has shown that there are probably two retrograde signals dependent on the tetrapyrrole pathway. In fact the two FLP isoforms are encoded by a single gene, whose messenger is alternatively spliced, the production of the shorter or of the longer form is under control of signals from the chloroplast and from the light perception (Falciatore et al., 2005).
The retrograde signal dependent on the tetrapyrrole pathway has been investigated also in higher plants. In the early ‘90s a genetic screen allowed for the isolation of 5 genome uncoupled mutants (GUN). The approach was based on the repression of the expression of photosynthetic genes in seedling treated with Norfluorazon (NF), a herbicide that blocks the synthesis of carotenoids. The gun mutants were expressing a reporter gene under the control of the Lhcb promoter even if treated with NF (Susek et al., 1993). Four of the gun mutants characterized present lesions in genes involved in the tetrapyrrole biosynthetic pathway. Two of them, gun2 and gun3, encode the enzymes heme oxygenase and phytochromobilin synthase that are necessary to produce phytochromobilin from heme. In their absence the cells accumulate heme, which inhibits the GluTR, the first enzyme of the tetrapyrrole pathway (see previous chapter on the regulation of chlorophyll biosynthesis) (Pontoppidan and Kannangara, 1994;
Vothknecht et al., 1998). The gun5 mutant is affected in the gene for the H subunit of the MgCh and gun4 had a mutation in a protein that binds to the MgCh to increase the kinetics of the reaction (Larkin et al., 2003; Mochizuki et al., 2001). The common phenotype of these four mutants is a decrease in the concentration of the chlorophyll precursors below the branching point of the tetrapyrrole pathway (Mochizuki et al., 2001; Strand et al., 2003). It has been proposed that accumulation of Mg-protoporphyrin IX may cause the repression of nuclear genes in the dark or during NF treatment in the light in seedlings (Strand et al., 2003). Evidence of the presence of Mg-protoporphyrin IX in the cytosol of NF treated wild type seedling (Ankele et al., 2007) led to the hypothesis that in certain conditions Mg-Protoporphyrin IX can
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exit the chloroplast to activate nuclear factors that are responsible of the repression of a set of nucleus-encoded chloroplastic proteins (Nott et al., 2006; Strand, 2004; Strand et al., 2003).
The characterization of an Arabidopsis line knocked-out for the Mg-protoporphyrin methytransferase seemed to confirm this model (Pontier et al., 2007). Recently, more precise analysis of the chlorophyll precursor concentration in seedlings treated with NF challenge the role of Mg-protoporphyrin IX. Two groups independently report that in NF treated seedlings exposed to light, the Mg-protoporphyrin IX concentration was lower than in untreated samples, and that accumulation of this tetrapyrrole is not sufficient to repress the LHCB mRNA accumulation in gun mutants (Mochizuki et al., 2008; Moulin et al., 2008). Another discrepancy between the experimental data and the role proposed for the Mg-protoporphyrin IX in retrograde signaling is the fact that mutants affected in the CHLI subunit of MgCh do not seem to have a gun phenotype, even if the enzyme activity is impaired like for the mutants of the other two subunits (Mochizuki et al., 2001).
In conclusion it is now clear that the tetrapyrrole biosynthetic pathway is a source of retrograde signals. One of the main open questions is the identification of the actual signal. The accumulation of Mg-protoporphyrin IX by itself does not seem to be sufficient.
Another evidence against the model proposed is the high toxicity of the tetrapyrroles. In fact accumulation of these compounds may lead to a rapid photo-oxidation of essential cellular components that may cause cell death. It is possible that one of the enzymes of the pathway is by itself capable of triggering a signal to repress nuclear transcription. This role has been proposed for the CHLH subunit of the MgCh, for the GUN4 protein and for the Mg- protoporphyrin IX methyltransferase (Larkin et al., 2003; Nott et al., 2006; Pontier et al., 2007).
Interestingly it has been recently shown that porphyrins promote the association of the MgCh and the GUN4 protein with the chloroplastic membrane, a mechanism that may favor the Mg branch versus the Fe branch of the tetrapyrrole pathway as the protoporphyrinogen IX oxidase,
35 the enzyme that produces the protoporphyrin IX, is membrane associated (Adhikari et al., 2009). The association of the complex with the membrane may also be important to transmit a signal to the cytosol.
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I I. Articles and Results
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Article 1: The FLP proteins act as regulators of chlorophyll synthesis in response to light and plastid signals in Chlamydomonas
In this work we showed that the FLP proteins of Chlamydomonas reinhardtii are the orthologues of the Arabidopsis thaliana Flu protein, a negative regulator of chlorophyll synthesis.
In the absence of Flu, Arabidopsis accumulates high levels of protochlorophyllide and upon illumination it bleaches and dies. We reduced the level of FLP in Chlamydomonas by siRNA and we could observe a similar phenotype.
Interestingly the two isoforms of FLP are produced by alternative splicing of the primary transcript of a single gene. The splicing and the expression of the gene are regulated by light and by plastid signals. We showed that in mutants accumulating chlorophyll precursors, the FLP gene is overexpressed in the dark. Moreover we observed that the presence of specific chlorophyll precursors can induce the expression of a specific isoform of the protein.
We could correlate the presence of the short form to the accumulation of precursors common also to the heme branch of the tetrapyrrole pathway. Instead the long form was expressed in mutants accumulating precursors of the magnesium branch of the pathway. We conclude that the FLP gene is regulated by two different retrograde signals, one inducing the expression of the gene, and the other promoting alternative splicing.
Personal contributions to the study:
• Establishment of a method to quantify the chlorophyll precursors in the laboratory
• Quantification of the pigments in figure 6, figure 7, figure 8 and supplementary tables
• Participation in the analysis of the greening of y1 mutant
• Participation in the analysis of the siRNA clones
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Search for proteins involved in FLP regulation
Genetic approach
In order to identify proteins involved in FLP gene expression in response to chloroplastic signals we tried a genetic approach. I cloned the FLP promoter upstream of the genes coding for the arylsulfatase (Davies et al., 1992) or the luciferase from Renilla reniformis reconstructed with the codon usage of Chlamydomonas (Fuhrmann et al., 2004). The idea was to screen for mutants in which these reporter genes are misexpressed. Unfortunately I was not able to observe the expression of these two genes under the control of the FLP promoter which is apparently too weak for these assays.
I tried a similar approach to identify factors involved in the regulation of the alternative splicing of the FLP hnRNA. In this case I constructed a chimeric gene fusing the 5’ part of the FLP gene (up to the 4th exon) to the luciferase reporter gene (Fuhrmann et al., 2004) under the control of the strong Hsp70-RbcS2 promoter. I then produced two versions introducing a stop codon in the 3rd exon or a stop codon in the 4th exon and a compensatory mutation in the 3rd exon. The two constructs were supposed to express the luciferase reporter only if the 3rd exon was excluded or included in the mature messenger, respectively. All attempts to detect the expression of these constructs were unsuccessful.
Biochemical approach: Experimental design
Expression of the FLP gene is regulated by alternative splicing which is controlled by retrograde signals from the chloroplast (Falciatore et al., 2005). In order to identify factors involved in the regulation of the splicing event we decided to take a biochemical approach. The experimental approach involved an enrichment of soluble proteins of Chlamydomonas binding to nucleic acids with heparin-conjugated beads. The fractions obtained were then tested for their
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ability to bind to the Flp RNA by electrophoretic mobility shift assays (EMSA). The fractions with specific RNA binding activity were pooled together and further enriched for proteins binding to the FLP pre-mRNA by GRNA chromatography (Czaplinski et al., 2005). Purified proteins were then separated by SDS-page gel electrophoresis and visualized by silver staining.
Specific bands were cut from the gel and analyzed by mass spectrometry (Figure1).
Wild type in continuous light expresses mainly the short isoforms of FLP, whereas the pc1 and the y1 mutants kept in the dark express mainly the long form (Falciatore et al., 2005). The y1 mutant is a yellow-in-the-dark strain which lacks a functional photosynthetic apparatus in the dark and this can trigger other retrograde signals not related to the tetrapyrrole pathway. The pc1 mutant on the other hand is affected in the light-dependent protochlorophyllide reductase and assembles functional photosynthetic complexes in the dark. Thus we decided to compare the extracts of a wild-type strain grown in continuous light and of a pc1 mutant grown in the dark.
Figure1. Scheme of the experimental strategy used to identify factors involved in the alternative splicing of the FLP pre-mRNA. Total soluble extracts were obtained from dark grown pc1 cells and WT cells grown in continuous light. Heparin-agarose beads were used to enrich for nucleic acids binding proteins and checked by electrophoretic mobility shift assays (EMSA) for binding
57 to FLP pre-mRNA. Positive fractions were subjected to GRNA chromatography and eluates were separated by SDS-page polyacrylamide gel electrophoresis. Specific bands were sequenced by mass spectrometry.
Enrichment for nucleic acid binding proteins from total soluble extract
Total soluble extracts were obtained from dark-grown pc1 cells and wt cells grown in continuous light. The extracts were loaded on heparin-conjugated beads, eluted with a step gradient of potassium acetate and dialyzed against GRNA Buffer (See methods). Heparin- agarose fractionation has been used successfully in Chlamydomonas to isolate specific RNA binding factors (Danon and Mayfield, 1991; Yohn et al., 1998). We decided to use this method as a first step in order to enrich the total soluble extracts for nucleic acid-binding proteins. The concentration of the dialyzed fractions was estimated by SDS-PAGE followed by Coomassie staining (not shown). The fraction at 1.6 M potassium acetate contained less proteins compared to the other ones, probably because the major part of the proteins were already eluted. For the following steps we used the maximum volume allowed for this fraction and an equal amount of proteins for the others. Samples were verified for specific binding activity by EMSA with a RNA arising from the 3rd exon of the FLP gene (Flpi23i) with 55 nucleotides of intronic sequence on the 5’ and the 3’ ends (Figure 2A).
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Figure 2. Panel A: schematic view of the FLP gene, exons are depicted as full boxes and numbered. Constructs used in the electromobility shift assay and in the GRNA chromatography are depicted: Flpi23i contains the 3rd exon and 55 nucleotides of intron sequences on both ends, Flpe11e contains 123 nucleotides from exon 1. Three fmol of probe were used in each lane. Panels B and C: EMSA of the Flpi23i RNA with the fractions obtained by heparin- agarose fractionation. Panel B without competitor; panel C with 100 fold excess of non labeled Flpi23i as competitor.
