Prof. Dr. Michael Schroda

Group "Plant Molecular Chaperone Networks and Stress"
New Position: Professor of Molecular Biotechnology at the University of Kaiserslautern. Head of the department.

Using the unicellular green alga Chlamydomonas reinhardtii as model organism, we are working on four main projects:

  • Functional investigation of the chloroplast HSP70/HSP90 chaperones
  • Investigation of the mechanisms by which the HSP70A promoter activates transgene expression
  • Dissection of the sterss response in Chlamydomonas
  • GoForsys

1. Functional investigation of the chloroplast HSP70/HSP90 chaperones

Rationale

Chaperones are a specialized class of proteins, whose most well-known function is to help other proteins to assume and maintain the native folding state. In addition, chaperones participate in numerous other activities like the translocation of proteins across biomembranes, protein complex assembly/disassembly, maturation of signal transduction components, tagging of unfoldable proteins for degradation, etc. While chaperones have been extensively studied in bacteria, mitochondria, the ER, and the eukaryotic cytosol, comparably little is known about their activities in chloroplasts. This is surprising, since chloroplasts harbour a unique compartiment which is the ultimate energy source for almost all life on earth - the thylakoid membranes. Moreover, in light of the growing importance of the chloroplast as the compartment of choice for the expression of recombinant proteins an understanding of its protein folding machinery is indispensable.

Aim

To biochemically characterize the chloroplast chaperone networks and to elucidate their roles in chloroplast biogenesis and maintenance of chloroplast functions.

Current State

We have identified and biochemically characterized several components of the chloroplast HSP70 and HSP90 chaperone systems in Chlamydomonas. Moreover, we have gained insights into the regulation of HSP70B’s chaperone activity and identified one of its substrates. The current state of knowledge is summarized below:
Click the image to see a larger version with description.

Currently addressed questions and approaches

  • Which are the substrates of the chloroplast HSP70 and HSP90 systems?
    → We apply QUICK-X, which by a combination of RNAi, stable isotope labelling, immunoprecipitation, and quantitative mass spectrometry allows for the identification of protein-protein interactions at high sensitivity.
  • How does HEP2 activate HSP70B after import into the chloroplast?
    → We generate homology models of HSP70B and HEP2 and simulate their interactions by the RosettaDock program. Moreover, we crosslink recombinant HSP70B and HEP2 and identify dipeptides by mass spectrometry. Deduced interaction surfaces are verified by site-directed mutagenesis.
  • Why do the HSP90/HSP70 systems interact with VIPP1?
    → We investigate the function of VIPP1 in the chloroplast by analysing VIPP1-RNAi strains at the levels of physiology and proteome composition using quantitative mass spectrometry. Moreover, we seek for more VIPP1 interacting proteins with QUICK-X.
  • Is HSP70B redox-regulated?
    → We investigate the chaperone activity of HSP70B in vitro under certain redox states. Moreover, we mutate the redox-active cysteines in HSP70B and analyse effects of redox stress in vitro and in vivo

2. Investigation of the mechanisms by which the HSP70A promoter activates transgene expression

Rationale

Transgene silencing is frequently observed in eukaryotic systems and may be mediated by epigenetic mechanisms. This is particularly the case in Chlamydomonas and therefore epigenetic (trans)gene silencing is easy to study in this organism. Approaches undertaken so far aimed only at the identification of factors involved in transgene silencing, whereas no information exists on factors involved in transgene activation. We have in earlier work found that transgene expression in Chlamydomonas becomes activated when the promoter of the HSP70A gene is fused upstream from transgene-driving promoters. Hence, the mechanism underlying HSP70A promoter-mediated transgene activation might be applied to generally facilitate transgenic approaches in eukaryotes.

Aim

To understand the molecular mechanisms by which the HSP70A promoter activates transgene expression.

Current state

We could show that the transgene activating effect is mediated largely by heat shock elements, apparently via heat shock transcription factors (HSFs), as outlined in the following Figure:
Click the image to see a larger version with description.

Currently addressed questions and approaches

  • Which factors(s) recruited by HSF carry out chromatin remodelling?
    → We apply QUICK-X, which by a combination of RNAi, stable isotope labelling, immunoprecipitation, and quantitative mass spectrometry allows for the identification of protein-protein interactions at high sensitivity.
  • Which epigenetic marks are associated with transgene activation by the HSP70A promoter?
    → We use Chromatin Immunoprecipitation (ChIP), for which we have established a robust protocol for Chlamydomonas

3. Dissection of the stress response in Chlamydomonas

Rationale

The insufficient adaptation of many crop plants to environmental stress has a significant impact on crop yield. This problem might be solved by transgenic approaches, given that the underlying mechanisms of protection to stress were understood. A large part of the plant’s stress response is mediated by heat shock transcription factors (HSFs). However, the high complexity of the HSF family in higher plants with at least 21 members has complicated the dissection of the stress response in plants. In contrast, the presence in Chlamydomonas of only one canonical HSF that exhibits all characteristic features of plant class A HSFs, made it attractive to use this alga for studying fundamental principles of the plant stress response.

Aim

To understand the fundamental principles of the heat shock factor dependent stress response in Chlamydomonas

Current state

We have combined pharmaceutical and antisense/RNAi approaches, which have enabled us to establish a working model for the regulation of the stress response in Chlamydomonas (see figure below). In collaboration with Alexander Skupin and Oliver Ebenhöh at the MPI this model has been translated into a mathematical model, which is able to reproduce our data from inhibitor feeding.

Currently addressed questions and approaches

  • Is our model of the HSF1-dependent stress response correct?
    → Predictions by the mathematical model are tested experimentally using RNAi, qPCR, pharmaceutical, and quantitative mass spectrometry approaches
  • Which genes are regulated by HSF1?
    → In collaboration with Wolfgang Hess at the University of Freiburg we analyse expression profiles of HSF1-RNAi and wild-type strains using Microarrays

4. GoFORSYS

Rationale

Photosynthesis is a central determinant of crop growth and productivity, as well as being a crucial parameter in determining the distribution of species and, at an ecosystem level, being the major route by which the biosphere affects the composition of the atmosphere, with all of the implications for global change. The rate of photosynthesis will be a crucial contributor towards bio-fuels production, while maintaining capacity for food production. GoFORSYS is a Systems Biology approach to underpin the analysis and improvement of crop plant photosynthesis. Central to the subject is the comprehensive systems analysis of the expression and regulation of photosynthesis in response to selected environmental factors in a model algal system, i.e. Chlamydomonas reinhardtii, and the integration of the insights with research on a model higher plant and a model crop plant.

Aim

To investigate how plants adjust and optimize photosynthesis to a changing environment, with the ultimate goal of understanding how this translates into plant biomass accumulation and, hence, growth.

Current state

Chlamydomonas is grown photoautotrophically under controlled conditions in a bioreactor and a single parameter (e.g. light intensity) is changed. Adaptations of the cells to the changed parameter are then analyzed by the GoFORSYS consortium at the levels of nuclear and organellar transcripts, polysome loading, proteome, enzyme activities, metabolome, lipidome, and activities of the thylakoid membrane complexes. After integration of obtained data these are used to parametrize mathematical models generated before.

Our contribution to GoFORSYS

We are analyzing relative changes at the proteome level using quantitative mass spectrometry and in collaboration with Alexander Erban and Jachim Kopka at the MPI analyze changes in metabolite profiles.

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