Structure - function analysis of Arabidopsis thaliana CC-type glutaredoxins ROXY1 and ROXY9

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Electron transfer reactions play essential roles in all living organisms. The reduction of oxygen during respiration, for instance, is the basis for the evolution of the complex life that is shaping our planet. Although being essential for aerobic metabolism, oxygen also leads to unwanted oxidation of biomolecules. These can be reduced by components of the anti-oxidative system. Serving as essential components of this protective system, glutaredoxins (GRXs) catalyze the reduction of disulfide bonds. In addition or alternatively, some of them coordinate iron-sulfur (FeS) clusters and thus facilitate FeS cluster synthesis, transfer to target proteins and redox and iron sensing. GRXs containing variants of a CP(Y/F)C or a CGFS motif in the active site occur in all types of organisms. Intriguingly, a third type of GRXs - being characterized by a CC(M/L)(C/S) motif - is only found in the genome sequences of land plants. In contrast to CPYC- and CGFS-type GRXs, the biochemical and biophysical features of these land plant-specific CC-type GRXs are poorly described. A hallmark is their ability to interact with and regulate members of the TGACG-binding protein (TGA) family of transcription factors. This proposal asks the question whether CC-type glutaredoxins still require the functions of the evolutionary older canonical GRXs to regulate TGA activity. We focus on two of the 21 Arabidopsis thaliana glutaredoxins (ROXY1 and ROXY9), since they most likely regulate their cognate TGAs by different mechanisms. ROXY1 negatively regulates TGA factor PAN to determine the correct number of petals during flower development. ROXY9 can repress the activities of the redundant factors TGA1 and TGA4, which regulate growth and defense responses. Proteins with mutations in critical amino acids will be characterized with respect to their redox states, oxidoreductase activities, FeS cluster binding capacities and in planta functions. These analyses will show, which in vitro properties are required for the in vivo activities of ROXY1 and ROXY9.