La danza del Torbellino

Change of translation rates by redox levels as described for the translational repressor NAB1 does not only take place in the cytosol. Reversible formation of a disulfide bridge also influences psbD translation in the chloroplast of C. reinhardtii as it was shown in this thesis. Establishment of that disulfide bridge associates RBP40 to Nac2 for efficient D2 synthesis in the light (see 3.3: Schwarz, et al. 2011). The assembly of translational activators to a target

RNA is an important key player of organellar gene expression as translation is supposed to be the pacemaker of protein synthesis in the chloroplasts (Eberhard, et al. 2002). In a comparable manner redox-dependent interaction of several proteins involved in D1 synthesis was proposed (see 1.4.3, Barnes and Mayfield 2003, Somanchi, et al. 2005, Alergand, et al. 2006). This also holds true for the light dependence of the synthesis rates of the D1 and D2 proteins (Malnoë, et al. 1988, Trebitsh, et al. 2000, see 3.3: Schwarz, et al. 2011).

The redox regulation of the synthesis of the D2 protein presents a certain challenge. The disulfide bridge that is necessary for assembly of the Nac2/RBP40 complex and subsequent translation of psbD mRNA is formed in the light and opened in the dark. Lack of photosynthetic electron flow during the night cannot reduce this disulfide bridge, whereas during the day this covalent bond exists in presence of reducing equivalents that are produced from photosynthesis. It is shown in this thesis that a possible source in Chlamydomonas, supplying the required electrons for reduction of the Nac2-RBP40 disulfide bridge in the dark, could be the NADPH-dependent thioredoxin reductase class C (NTRC) system (see 3.3: Schwarz, et al. 2011), which is exclusive to photosynthetic organisms (Pascual, et al. 2011). In comparison to other NADPH-dependent thioredoxin reductases, NTRC contains an additional thioredoxin domain and offers an alternative electron pathway independent of light and ferredoxin. The thioredoxin is reduced by electrons from NADPH by using FAD as a co- factor (Chibani, et al. 2010). This alternative electron source is possible in the dark as NADPH can be generated by the oxidative pentose phosphate pathway (Neuhaus and Emes 2000). One of the described functions of this enzyme was the reduction of thiol-dependent peroxidases during stress responses in Arabidopsis (Moon, et al. 2006). NTRC mutants were hypersensitive to extended periods of darkness due to accumulation of hydrogen peroxide whereas electrons - required for peroxide reduction - can be provided by ferredoxin during the day (Pérez-Ruiz, et al. 2006). Other pathways affected by NTRC include functions during biogenesis of aromatic compounds and chlorophyll as well as protection against abiotic stress (Serrato, et al. 2004, Stenbaek, et al. 2008, Lepistö, et al. 2009). Moreover, it has been linked to the regulation of starch synthesis in combination with the ferredoxin/thioredoxin system (Ballicora, et al. 2000, Michalska, et al. 2009). Data provided here suggest an additional role of this multifunctional enzyme: the involvement in chloroplast gene expression in the dark. The finding of a possible regulatory function of NTRC in synthesis/assembly of PSII reinforces the linkage between intracellular transport, metabolism and nutrient availability by alteration of redox states and post-translational modifications (Geigenberger, et al. 2005,

Bräutigam, et al. 2009, see 3.4: Wobbe, et al. 2009, Balsera, et al. 2010, Dorn, et al. 2010, Blifernez, et al. 2011, see 3.3: Schwarz, et al. 2011). In conclusion, a further validation of the involvement of the NTRC system in D2 synthesis would provide a direct link between expression of chloroplast genes and carbon metabolism in C. reinhardtii. This would provide an important crosstalk between photosynthesis and catabolic cell activities. Further details that await elucidation include the identification of the cysteine residue of Nac2 interacting with RBP40, the elucidation of the function of a predicted NADPH binding site in Nac2 and the characterization of the electron acceptor necessary for the formation of the disulfide bridge between Nac2 and RBP40.

A further example of a chloroplast protein whose translation relies on the organellar redox state is the large subunit of Rubisco. Synthesis of that protein responds to shifts in the glutathione pool caused by oxidative stress (Irihimovitch and Shapira 2000). Oxidative events at thiol groups in the Rubisco protein induce a conformational change which leads to the exposure of an N-terminal structure with homologies to RRM domains and the human U1A splicing factor. This change of protein confirmation, possibly assisted by chloroplast chaperones of the HSP60 family, enables Rubisco to bind RNA unspecifically under oxidizing conditions (Hemmingsen, et al. 1988, Yosef, et al. 2004). Binding of the protein to RNAs in its vicinity, including rbcL mRNA, might stall protein synthesis to lessen the effects of oxidative stress. (Cohen, et al. 2005). Further results also showed a co-regulation of chaperones belonging to HSP70 complexes and stress conditions as well as their involvement in the synthesis of bacterial Rubisco (Checa and Viale 1997, Shrager, et al. 2003).

Additionally, HSP70 was found to be a potential thioredoxin target itself (Lemaire, et al. 2004). During this thesis, it was shown for members of the HSP70 chaperone family in Chlamydomonas that chaperone complexes themselves contain RNA. It was not elucidated so far if the interaction with RNA is direct or if the chaperone binds co/post-translationally to the protein encoded by that RNA (see 3.5: Dorn, et al. 2010). This gives rise to the opportunity that the organellar redox state directly regulates translational activity for plastidial proteins. As an alternative, translational regulation could occur via feedback mechanisms through disassembly of resulting protein complexes in response to the oxidation level of a cellular compartment. An example of translational regulation by the assembly state of the resulting complex is the synthesis of PSII core subunits in Chlamydomonas according to the CES principle, e. g. psbB translation also depends on redox-dependent D2 synthesis in addition to

the level of available chlorophyll (see 1.4.3, Eichacker, et al. 1992, Plumley and Schmidt 1995, Minai, et al. 2006).

These examples of post-transcriptional regulatory events for synthesis of proteins show that fluctuating interactions between several factors are necessary for maintaining cellular survival while adapting to external (e.g. light conditions) and internal aspects (e.g. available interaction partners).

In conclusion, the results of this thesis enhanced the understanding of several aspects that fulfill important functions in the post-transcriptional regulation of chloroplast gene expression in C. reinhardtii.

5. Appendix