Physcomitrella patens as a Model Organism in Plant Research
Choosing the right model organisms in research is crucial for addressing biological questions depending on diverse conditions and aspects. Arabidopsis thaliana (A. thaliana) is since decades still the most prominent and favoured plant organism in various research fields. However, more and more photosynthetic organisms are being used for research as there is ever increasing access to genome, transcriptome and proteome data.
For several moss species genomic and transcriptomic data are available including Ceratodon purpureus (Szövényi et al., 2015), Sphagnum (Shaw et al., 2016) and Funaria species (Chang et al., 2016; Article 1). The first and thus most developed and established moss model is Physcomitrella patens (P. patens), which belongs to the family Funariaceae. It spreads naturally in the temperate zones of North America and Europe (Cove et al., 2009a) and prefers littoral zones of lakes and rivers with damp soil. The advantageous phylogenetic position between algae and vascular plants makes P. patens an important model system for comparative genomic studies. Most laboratory strains originate from one single spore which was collected by Whitehouse in Gransden Wood, UK and cultivated by Engel in 1968 (Cove, 2005). The complete genome of P. patens was sequenced and annotated in 2008 (Rensing et al., 2008) and updated in 2013 (Zimmer et al., 2013), resulting in approximately 36 000 predicted gene models spread on 27 chromosomes (Reski et al., 1994). In addition to the improvement and expansion of existing information, the description of non-protein coding loci (including e.g. tRNA, rRNA and miRNA) as well as information concerning alternative splicing variants, are available. The resulting data are provided on cossmoss.org, the database for P. patens genome/transcriptome resources.
As in all plants, the life cycle of bryophytes comprises two phases: a haploid gametophytic and a diploid sporophytic. But in contrast to vascular plants, in which the diploid stage is the dominant one, bryophytes spend most of their life cycle in the haploid gametophytic stage (Reski, 1998). Thus mosses possess only one allele for each gene and are attractive for gene manipulation as time-consuming back crossings for homozyous plants are not required.
The life cycle of P. patens (Figure 1 in article 1) starts with the germination of a haploid spore and depending upon light and water availability, a filamentous and single-cell layer tissue, called protonema grows by apical cell division. Via bud formation the tissue performs a juvenile-to-adult transition in which during the formation of a three-faced apical cell, the gametophore with leaf-like structures arises (Reski, 1998). After a certain period of specific light conditions, water availability and temperature the sexual organs develop on top of the gametophore (Hohe et al., 2002), with P. patens being monoecious, bearing both reproductive organs on the same plant (Cove, 2005). The antheridia produce the male gametes equipped with flagella whereas the female gametes are produced in archegonia. The fertilisation is dependent on a water film, and from the resulting diploid zygote develops the sporophyte, which is physically linked to the gametophore. The sporogonium is located on the upper part of the sporophyte, containing about 5000 haploid spores produced after meiosis. A complete life cycle can be effected in approximately three months (Cove, 1992). The cultivation of P. patens in the laboratory is done under axenic conditions. The plant can be grown either on agar plates or as protonema tissue in liquid culture under standardized conditions. In contrast to higher plants, almost all cell types are able to regenerate into an adult moss plant (Reski, 1998), thus via frequently disrupting plant material the moss tissue can be cultivated continuously.
The Thioredoxin System in Plants
Land plants are sessile organism compared to other eukaryotes. They have to cope directly with the surrounding fluctuating environment possibly featuring stresses of abiotic or biotic nature, and are dependent on their fast reaction and adaption to new situations. The availability of water, temperature variations, light intensity and the attack of herbivores are only a few examples of potential stress conditions for plants. As a successful strategy to withstand these challenges, a large and complex redox network in plants has evolved. Plants possess in contrast to all other organisms the highest number of proteins/genes involved in redox signalling. Major stress signalling molecules in plants are reactive oxygen species (ROS) including superoxide anions (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-). These messengers are generated in a ―reasonable‖ amount constantly under normal conditions during photorespiration and oxidative protein folding, but highly accumulate during extreme environmental stress situations and can lead to DNA damage and subsequent cell death (Apel and Hirt, 2004). Therefore several detoxification systems distributed in several cell compartements have been evolved, e.g. superoxide dismutase, methionine sulfoxide reductases, glutathione peroxidase, peroxiredoxins. The reduction and activation of those are dependent on the TRX and GRX systems (Gelhaye et al., 2005).
Electron Donor Systems in Plants
Plants possess short bacterial-type NADPH-thioredoxin reductases (NTRs) without selenocysteine residues (Arnér and Holmgren, 2000) with a subunit molecular weight of about 35 kDa. Due to their complex structure with additional organelles (e.g. the chloroplast), the number of TRX reduction systems in plants goes up to four (Figure 3). The first crystal structure of an eukaryotic NTR was solved from A. thaliana (Dai et al., 1996). Two classical NTRs are found in plant genomes, the NTRA and B, both dual targeted to cytosol and mitochondria, with NTRB being most abundant in mitochondria (Gelhaye et al., 2005; Reichheld et al., 2005). However, they share a high amino acid identity (95%) and therefore a redundant function. Single mutants of each of the reductases in A. thaliana completely lack a phenotype, whereas the double mutant shows a wrinkled seed phenotype and growth delay (Reichheld et al., 2007). Overexpression lines of NTRA in A. thaliana show high oxidative stress tolerance compared to wild type (WT) and ntra-ko plants (Cha et al., 2014). But compared to mammalian NTRs they do not seem to be essential for plant survival (Reichheld et al., 2007). NTR and TRX isoforms are also found in the nucleus (Delorme-Hinoux et al., 2016). The two other reductant systems are localized in chloroplasts and are unique to photosynthetic organisms, the ferredoxin/thioredoxin system (FTR-system) and the unusual NTRC (Figure 3). The FTR obtains its electrons directly from ferredoxin (FDX), whereas the NTRC needs NADPH, generated via the oxidative pentose phosphate pathway (OPP) and the photosynthetic reactions.
Light-Dependent Redox Regulation in Chloroplasts
For photosynthetic organisms one essential survival factor is the availability of light which is used as energy source and is converted into chemical compounds in chloroplasts via the photosynthetic reactions that was traditionally split into light reactions and dark carbon fixation. But to prevent futile processes in cases of missing or fluctuating light the two reactions need to be connected in a regulatory way to adjust chloroplast function to changing light conditions. This regulatory system is maintained by post-translational redox modifications and different proteins and protein families are involved in this complex network in plastids (Schürmann and Buchanan, 2008).
The Ferredoxin/Thioredoxin System
About two decades after the initial discovery of the Calvin-Benson cycle it was reported that several involved enzymes show a light-dependent activation, and these findings led later to the description of the FTR system, the major pathway responsible for light sensitive regulation of a large set of target chloroplast proteins with various functions (Schürmann and Buchanan, 2008). This system consists in a redox cascade involving the photosystems (PSII and PSI) and the proteins FDX, FTR and chloroplast TRXs (Figure 3).
After illumination of the photosystem II (PSII) and absorption of photons, water molecules are split and electrons released to be transported within the thylakoid membrane. Thereby a proton gradient is built which is used by the ATP synthase to produce ATP. The electrons move from the plastoquinone pool to cytrochrome b6f and further down via plastocyanin to the PSI, and are after light excitation transferred to FDX.
FDXs are iron containing small proteins present in bacteria, plants and mammals which shuttle electrons. In bacteria FDX is involved notably in iron sulphur assembly (Yan et al., 2015) and in humans in steroidogenesis (Sheftel et al., 2010). In plastids FDX is located in the stroma, binds to the thylakoid membrane and plays an important role as electron acceptor from the photosynthetic electron transport chain. It is a small ubiquitous protein of about 11 kDa and harbours a single [2Fe-2S] cluster for distribution of reducing equivalents to a number of proteins involved in different reactions and metabolic pathways (e.g. amino acids, lipid, sulphur and nitrogen assimilation) (Zurbriggen et al., 2008). However, FDX proteins are not only found in photosynthetic active tissue but also in plant roots as in A. thaliana, active notably in sulphite reduction (Hanke et al., 2004). Its gene expression is highly decreased during stress conditions and iron starvation (Zurbriggen et al., 2008). One of the major acceptor proteins is ferredoxin-NADP+-oxidoreductase (FNR) responsiple for the photoreduction of of NADP+ into NADPH, H+ which is later used in the Calvin-Benson cycle reactions. Another protein partner is the oxidoreductase FTR, a major electron supplier for the plastid TRX family and thereby putatively a central enzyme in the redox regulation cascade in chloroplasts (Droux et al., 1987).
Two Light-Induced Phosphatases Function in Carbon Fixation
The light/dark reduction mechanisms of regulatory disulphide bonds in enzymes resulting in conformational changes affecting the catalytic properties of these proteins is widely found in chloroplasts. Frequently such chloroplast enzymes have other cellular counterparts, but with missing or different regulatory cysteine residues in the amino acid sequence (Michelet et al., 2013).
Several targets of the FTR system are involved in carbon fixation or directly function in the Calvin-Benson cycle. Thereby light-generated NADPH can be used for carbon fixation and futile energy expenditure is prevented. The Calvin-Benson cycle consists of 11 enzymes catalysing 13 reactions. These can be split into three parts, starting with the CO2 trapping by RuBisCO followed at the expense of ATP and NADPH by the reduction of 1,3-bisphosphateglycerate and the release of glyceraldehyde 3-phosphate (G3P) for sugar production. In the third and largest part the CO2 acceptor ribulose-1,5-bisphosphate is recycled out of G3P molecules and ATP. In this section the activity of the two phosphatases FBPase and SBPase is required during recycling. FBPase is a homotetramer catalysing the dephosphorylation of fructose-1,6-bisphosphate (FBP) to fructose-6-phosphate and was the first enzyme described to be reduced via the FTR system (Buchanan et al., 1967). This enzyme has a cytosolic counterpart function in gluconeogenesis pathway which is not light activated (Daie, 1993). The chloroplast enzyme contains insertion parts with three cysteine residues, two of them sensitive to redox, with a remaining basal activity in the oxidized form (Jacquot et al., 1997). The crystal structure was solved in the oxidized form for the pea chloroplast enzyme (Chiadmi, 1999) and in a pseudo-reduced form for the pig kidney enzyme (Villeret et al., 1995). The conformational change upon reduction does not affect directly the active site of the enzyme but most likely the binding site of the cofactor magnesium (Chueca et al., 2002). SBPase in contrast is essentially unique to the eukaryotic Calvin-Benson cycle and to chloroplasts although it is found in organisms possessing apicoplasts, vestiges of plastids and which have lost the capacity to perform photosynthesis along the evolution process (Minasov et al., 2013). The redox active cysteine residues were identified via site directed mutagenesis (Dunford et al., 1998) and the reaction catalysed splits phosphate from the substrate sedoheptulose-1,7-bisphosphate (SBP) to generate sedoheptulose-7-phosphate. Both phosphatases, FBPase and SBPase, share a number of similar features and properties. They exhibit rather high homology in some portions of their amino acid sequences (Raines et al., 1992) even though the redox cysteines are located differently for each protein sequence. Besides reduction, their activity is dependent on the pH and level of cofactor magnesium present in the stroma. The substrates of their hydrolysis reactions, FBP and SBP, differ in size only by one carbon. However, SBPase is completely inactive in the oxidized state and requires light induction. In some cyanobacteria a bifunctional enzyme possessing both phosphatase activities was described (Tamoi et al., 1996), but its amino acid sequence is apparently not or very poorly related to the single phosphatases from eukaryotic organisms (Feng et al., 2014). FBPase and SBPase play important and essential key roles in the Calvin-Benson cycle reactions and are limiting steps of carbon fixation. Especially the reduction of SBPase expression levels in transgenic plants resulted in significant effects on plant growth and development with decrease of plant yield concerning shoot, leaf and floral biomass and vice versa (Lefebvre et al., 2005; Liu et al., 2012). Thus SBPase is a major determinant and a bottleneck of carbon assimilation rate and can directly be linked to photosynthetic capacity which makes the enzyme an interesting target in agriculture and breeding research (Raines, 2003).
Table of contents :
1.1 The Influence of Mosses on their Environment
1.1.1 Article 1: Can Mosses Serve as Model Organisms for Forest Research?
1.1.2 Physcomitrella patens as a Model Organism in Plant Research
1.2 The Thioredoxin System
1.2.1 The Thioredoxin System in Plants
1.2.2 Electron Donor Systems in Plants
1.2.3 The Thioredoxin Family in Plants
1.3 Light-Dependent Redox Regulation in Chloroplasts
1.3.1 The Ferredoxin/Thioredoxin System
1.3.2 Plastid Thioredoxins
1.3.3 Two Light-Induced Phosphatases Function in Carbon Fixation
1.1 Aim of the Thesis
2. Material and Methods
2.1 Chemicals, Media, Oligonucleotides and Enzymes
2.2 Cell Culture Conditions for P. patens
2.3 Generation of Transgenic Moss Lines
2.3.1 Transformation of P. patens
2.4 Molecular Biology
2.4.1 RNA Extraction
2.4.2 DNA Digestion
2.4.3 cDNA Synthesis
2.4.4 Polymerase Chain Reactions and Oligonucleotides
2.4.5 Agarose Gel Electrophoresis
2.4.6 DNA Gel Extraction
2.4.7 Preparation of Plasmid DNA
2.4.8 DNA Precipitation with Ethanol
2.4.10 Bacterial Cultivation and Transformation
2.5 Analysis of FTR Mutant Lines
2.5.1 Protonema Growth Rate Determination
2.5.2 Light Treatments
2.5.3 Starch Accumulation
2.5.4 Net Photosynthetic Rate
2.5.5 Pulse Amplitude Modulation (PAM) Measurements
2.5.6 roGFP2 Measurements and Analysis
2.6.1 Protein Purification from E. coli
2.6.2 SDS-Page Gel
2.6.3 Crystallization and Structure Determination
2.6.4 Enzyme Activation and Reduction Assays
3. Results and Discussion
3.1 Composition of Redox Regulation Member Proteins in P. patens
3.2 Characterization of FTR mutants in P. patens
3.2.1 Expression and Homology of the two FTR Genes in P. patens
3.2.2 Cloning Strategy for Generating FTR Mutant Lines
3.2.3 Growth Analysis under Different Light Treatments
3.2.4 Starch Accumulation during Various Light Conditions
3.2.5 CO2 Assimilation Rate and Photosynthetic Parameters
3.2.6 Ratiometric Analysis of Redox Sensitive roGFP2
3.3 Identification and Characterization of Unusual f-type TRXs in P. patens
3.3.1 Phylogenetic Analysis of TRX f-type Proteins
3.3.2 Reduction Capability of PpTRX f1 and PpTRX f2 show similar results
3.3.3 Different Electron Donors for PpTRX f1 and PpTRX f2
3.4 Biochemical and Structural Analysis of FBPase and SBPase from P. patens and Evolutionary Considerations of Redox Regulation in Chloroplasts
3.4.1 Article 2: Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories
3.4.2 Article 3: Dithiol disulphide exchange in redox regulation of chloroplast enzymes in response to evolutionary and structural constraints
7. Lists of Figures and Tables
8. Supplementary Data