Prof. Michael Hippler
Institute of Plant Biochemistry and Biotechnology
The University of Munster, Germany
Phone: +49 251 83 24790
Fax: +49 251 83 28371
Current Research Interests
Our research is aimed to understand plant cell responses and adaptation strategies to environmental stresses. Furthermore we aim to elucidate molecular mechanisms which are involved in assembly, function, maintenance and regulation of the photosynthetic machinery in oxygenic chloroplast photosynthesis. For this purpose, we are mainly using the green alga Chlamydomonas reinhardtii as a model system and combining molecular techniques like reverse genetics and proteomics to elucidate these processes. Hereby, we are engaged in different research lines.
(i) We are currently investigating the adaptation and remodeling of the photosynthetic apparatus as a response to iron-deficiency. We are also interested in an understanding of chloroplast iron-homeostasis as a whole. To gain insights into chloroplast iron-homeostasis we are carrying out a comparative proteomic approach and take advantage of stable isotope labeling by amino acids in Chlamydomonas (Naumann et al., 2007; Naumann et al., 2005) The investigation of putative candidate gene products in plastid iron homeostasis is done by reverse genetics using an RNAi approach as described by Cerutti and colleagues (Rohr et al., 2004). This approach was already successfully employed in our laboratory to suppress expression levels of Lhca3 (Naumann et al., 2005) and ferritin (Busch et al., 2008). Momentarily a focus is on the analysis of PGRL1 in iron-deficiency responses. In an RNAi strain, which accumulates lower PGRL1 levels in both Fe –replete and –starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in Fe sufficient conditions is increased, and the efficiency of cyclic electron transfer (CEF) under iron-deprivation is diminished. Furthermore, pgrl1-kd cells exhibit iron-deficiency symptoms at higher Fe concentrations than does the wild-type, although the cells are not more depleted in cellular iron relative to the wild-type as measured by mass spectrometry. Thiol trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may interconnect iron metabolism and CEF and thereby partitioning between linear photosynthetic electron transfer and CEF. We propose therefore that PGRL1 in C. reinhardtii possesses a dual function in iron sensing as well as modulation of cyclic electron transfer (Petroutsos et al., 2009). Future experiments will be directed to elucidate protein-protein-interactions of PGRL1 and metal-binding dynamics of the protein.
(ii) Light is a necessary substrate for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the time scale of seconds to minutes and involve feedback regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in stark contrast to flowering plants. Data that have been obtained in cooperation the group of Prof. K. Niyogi show that Chlamydomonas reinhardtii mutants diminished or -deficient in qEhave an impact in the expression of lhcsr genes, either due to RNAi-technology or due to knock-down of two of the three genes encoding LHCSR (formerly called LI818) (Peers et al., 2009). This protein is an ancient member of the light-harvesting complex superfamily, and orthologs are found throughout photosynthetic eukaryote taxa, except red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth in high light. LHCSR-RNAi or knock-out mutants have a reduced fitness in a shifting light environment compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Therefore the data indicate that plants and algae employ different proteins to dissipate harmful excess energy and protect the photosynthetic apparatus from damage. In the future insight into the NPQ mechanism in algae has to be gathered to understand the evolution of light management in algae and vascular plants.
(iii) At present we are also studying the remodeling of Chlamydomonas metabolism after transition to anaerobic growth conditions. The versatile metabolism of the green alga Chlamydomonas reinhardtii is reflected in its ability to acclimate to anaerobic conditions. The complex anaerobic response is also remarkable in the context of renewable energy since C. reinhardtii is able to produce hydrogen under anaerobic conditions. To identify proteins involved during anaerobic acclimation as well as to localize proteins and pathways to the powerhouses of the cell, chloroplasts and mitochondria from C. reinhardtii in aerobic and anaerobic (induced by 8 hours of argon bubbling) conditions were isolated and analysed using comparative proteomics. A total of 2315 proteins were identified. Further analysis based on spectral counting localised 895 of these proteins to the chloroplast, including many proteins of the fermentative metabolism. Comparative quantitative analyses were performed with the chloroplast-localised proteins using stable isotopic labeling of amino acids (13C6 Arginine/12C6 Arginine in an Arginine-auxotrophic strain). The quantitative data confirmed proteins previously characterized as induced at the transcript level as well as identifying several new proteins of unknown function induced under anaerobic conditions. These proteins of unknown function provide new candidates for further investigations taking advantage of reverse genetics. An important aim hereby is to understand the anaerobic response in the green alga. Another important aim will be to engineer Chlamydomonas strains that produce hydrogen in amounts allowing commercial exploration of the process.
(iv) In plant cells, the increase in cytoplasmic calcium [Ca2+]cyt represents a frequent response to various abiotic and biotic stresses. After changes in cytosolic Ca2+ signatures have been sensed, phoshorylation cascades appear to represent general downstream responses in Ca-dependent signal transduction. To elucidate [Ca2+]cyt dependent protein phosphorylation, identify [Ca2+]cyt dependent phosphoproteins, map phosphorylation sites and quantify phosphorylation, we are applying a phosphoproteomics approach, in which Arabidopsis thaliana and Chlamydomonas reinhardtii are taken as model systems.
(v) In another research line we are aiming to explore electron transfer and binding mechanisms between the soluble electron transfer proteins plastocyanin or cytochrome c6 and photosystem I (PSI) (Finazzi et al., 2005; Sommer et al., 2004; Sommer et al., 2006; Sommer et al., 2002). Additionally we aim to investigate the molecular recognition between PSI and its light-harvesting protein complex (LHCI), allowing the formation of the PSI/LHCI complex, subunit remodeling as well as efficient excitation energy transfer (Naumann et al., 2005; Nield et al., 2004; Storf et al., 2004; Takahashi et al., 2004). Hereby we are also taking advantage of absolute quantitation by using proteotypic peptide probes to enumerate the stochiometry of LHCI subunits per PSI core complex (Stauber et al., 2009).
(vi) We are establishing bioinformatics tools that are aimed to facilitate mass spectrometric analyses of eukaryotic organisms with complex gene structures. Here we are aiming to use mass spectrometric data for genomic data mining and annotation of protein-coding genes. We devised an algorithm, the GenomicPeptideFinder (GPF), which takes advantage of de novo amino acid predictions of MS/MS data enabling detection of intron-split and/or alternatively spliced peptides when deduced from genomic DNA (Allmer et al., 2004). We generated a platform where high throughput de novo sequencing of MS/MS spectra is combined with GPF performance operating on a computer cluster to analyse Chlamydomonas protein data (Allmer et al., 2006). We further developed a database for storage, analysis, presentation, and retrieval of information from mass spectrometric experiments (Allmer et al., 2008). Furthermore we devised an algorithm for evaluating quantitative mass spectrometric data stemming from SILAC or 15N-labeling (Specht et al., unpublished) experiments.
For proteomic analyses the laboratory is equipped with a hybrid linear ion-trap mass spectrometer (LTQ-Orbitrap, Thermo Finnigan) that is coupled to an UltiMate Nano liquid chromatography system (Dionex).
- Allmer, J., Kuhlgert, S. and Hippler, M. (2008) 2DB: a Proteomics database for storage, analysis, presentation, and retrieval of information from mass spectrometric experiments. BMC Bioinformatics, 9, 302.
- Allmer, J., Markert, C., Stauber, E.J. and Hippler, M. (2004) A new approach that allows identification of intron-split peptides from mass spectrometric data in genomic databases. FEBS Lett, 562, 202-206.
- Allmer, J., Naumann, B., Markert, C., Zhang, M. and Hippler, M. (2006) Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtii. Proteomics, 6, 6207-6220.
- Busch, A., Rimbauld, B., Naumann, B., Rensch, S. and Hippler, M. (2008) Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimises photo-oxidative stress in response to iron availability in Chlamydomonas reinhardtii. Plant J, 55, 201-211.
- Finazzi, G., Sommer, F. and Hippler, M. (2005) Release of oxidized plastocyanin from photosystem I limits electron transfer between photosystem I and cytochrome b6f complex in vivo. Proc Natl Acad Sci U S A, 102, 7031-7036.
- Naumann, B., Busch, A., Allmer, J., Ostendorf, E., Zeller, M., Kirchhoff, H. and Hippler, M. (2007) Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii. Proteomics, 7, 3964-3979.
- Naumann, B., Stauber, E.J., Busch, A., Sommer, F. and Hippler, M. (2005) N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii. J Biol Chem, 280, 20431-20441.
- Nield, J., Redding, K. and Hippler, M. (2004) Remodeling of light-harvesting protein complexes in chlamydomonas in response to environmental changes. Eukaryot Cell, 3, 1370-1380.
- Peers, G., Truong, T.B., Ostendorf, E., Busch, A., Elrad, D., Grossman, A., Hippler, M. and Niyogi, K.K. (2009) An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature, 462, 518-512.
- Petroutsos, D., Terauchi, A.M., Busch, A., Hirschmann, I., Merchant, S.S., Finazzi, G. and Hippler, M. (2009) PGRL1 participates in iron-induced remodeling of the photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii. J Biol Chem, 284, 32770-32781.
- Rohr, J., Sarkar, N., Balenger, S., Jeong, B.R. and Cerutti, H. (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J, 40, 611-621.
- Sommer, F., Drepper, F., Haehnel, W. and Hippler, M. (2004) The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6. J Biol Chem, 279, 20009-20017.
- Sommer, F., Drepper, F., Haehnel, W. and Hippler, M. (2006) Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry. J Biol Chem, 281, 35097-35103.
- Sommer, F., Drepper, F. and Hippler, M. (2002) The luminal helix l of PsaB is essential for recognition of plastocyanin or cytochrome c6 and fast electron transfer to photosystem I in Chlamydomonas reinhardtii. J Biol Chem, 277, 6573-6581.
- Stauber, E.J., Busch, A., Naumann, B., Svatos, A. and Hippler, M. (2009) Proteotypic profiling of LHCI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex. Proteomics, 9, 398-408.
- Storf, S., Stauber, E.J., Hippler, M. and Schmid, V.H. (2004) Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum). Biochemistry, 43, 9214-9224.
- Takahashi, Y., Yasui, T.A., Stauber, E.J. and Hippler, M. (2004) Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii. Biochemistry, 43, 7816-7823.