Functional analysis of the Arabidopsis thaliana glutaredoxin ROXY9 Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Microbiology and Biochemistry of the Georg-August University School of Science (GAUSS) submitted by Katrin Treffon from Radolfzell (Bodensee) Göttingen 2019 1 2 Thesis Committee Prof. Dr. Christiane Gatz, Department for Plant Molecular Biology and Physiology, Albrecht-von-Haller-Institut, Georg-August-Universität Göttingen Prof. Dr. Ivo Feußner, Department for Plant Biochemistry, Albrecht-von- Haller-Institut, Georg-August-Universität Göttingen Dr. Marcel Wiermer, Department for Molecular Biology of Plant-Microbe Interactions, Albrecht-von-Haller-Institut, Georg-August-Unviersität Göttingen Members of the Examination Board Referee Prof. Dr. Christiane Gatz, Department for Plant Molecular Biology and Physiology, Albrecht-von-Haller-Institut, Georg-August-Universität Göttingen 2nd Referee Prof. Dr. Ivo Feußner, Department for Plant Biochemistry, Albrecht-von- Haller-Institut, Georg-August-Universität Göttingen Further members of the Examination Board Prof. Dr. Andrea Polle, Department for Forest Botany and Tree Physiology, Büsgen-Institut, Georg-August-Universität Göttingen Prof. Dr. Volker Lipka, Department for Plant Cell Biology, Albrecht-von- Haller-Institut, Georg-August-Universität Göttingen PD Dr. Thomas Teichmann, Department for Plant Cell Biology, Albrecht-von- Haller-Institut, Georg-August-Universität Göttingen Date of oral examination: 25.03.2019 3 Declaration I hereby declare that I prepared the dissertation entitled “Functional analysis of the Arabidopsis thaliana glutaredoxin ROXY9” independently and without any unauthorized help. I confirm that I did not apply for a Ph. D. or Dr. rer. nat. at any other University. Neither the entire dissertation nor parts of this dissertation have been presented to another examination board. Katrin Treffon Göttingen, 08.02.2019 4 Summary _____________________________________________________________________ Glutaredoxins are nearly ubiquitious small enzymes which protect the thiol groups of cellular proteins from oxidative stress, contribute to iron-sulfur cluster biogenesis or regulate protein activities via redox modification. Three different classes of glutatreoxins are distinguished by their active site motif: CPYC-type glutaredoxins catalyze the reduction and oxidation of thiol groups and mediate defense against oxidative stress; CGFS-type glutaredoxins are weak catalysts but associate with iron-sulfur clusters, possibly transferring them to target proteins. Whereas CPYC- and CGFS-type glutaredoxins are found in all types of organisms, CC-type glutaredoxins, which are called ROXYs in Arabidopsis thaliana, are restricted to land plants. In contrast to their relatively well characterized relatives, CC-type glutaredoxins are biochemically poorly understood. Information about these glutaredoxins is only available from in vivo studies. Mutant analysis and studies with plants ectopically expressing ROXYs have shown that they modulate TGACG binding (TGA) factor activity. As an example, the CC-type glutaredoxin ROXY9 was shown to repress TGA1- mediated hyponastic growth when overexpressed. Since TGA1 was shown to be redox-regulated in plants treated with the defense hormone salicylic acid, it was speculated that ROXY9 represses TGA1 via catalytic activity. Therefore, this study was set up to analyze whether recombinant ROXY9 exhibits oxidoreductase and/or iron-sulfur cluster binding activity. Up to now, difficulties in the purification of CC-type glutaredoxins has been the bottleneck for their biochemical characterization. Similarly, ROXY9 could not be purified in a soluble, non-aggregated state when using Escherichia coli as an expression host. Strikingly, expression in insect cells resulted in large amounts of monomeric and soluble ROXY9 fused to a strep-MBP-tag. However, the protein turned out to oxidize quickly under aerobic conditions. Reduction of the oxidized protein by glutathione might be inefficient, which could explain the inactivity of ROXY9 towards the typical glutaredoxin substrates bis(2- hydroxyethyl)disulphide (HEDS), insulin, and the redox-sensitive green fluorescent protein (roGFP). Still, a weak reductase activity of ROXY9 towards glutathionylated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was observed, suggesting that ROXY9 in principle can reduce target proteins. In contrast to its weak reductase activity, ROXY9 turned out to glutathionylate roGFP efficiently with the help of glutathione disulfide, suggesting that 5 ROXY9 might repress TGA1 in vivo via glutathionylation. However, because all these observations might have been influenced by the oxidation of the protein, the catalytic activity of ROXY9 has to be reproduced with fully reduced protein. Aside from catalysis, ROXY9 appeared to bind an iron-sulfur cluster under anaerobic conditions. This raises the alternative hypothesis that it could repress TGA1 via recruitment of an iron-sulfur cluster-dependent repression complex. In vivo analysis of the repression capacity of overexpressed mutated ROXY9 versions suggested that ROXY9 requires the second cysteine and the tyrosine of its extended active site motif CCLCY for its activity. Future experiments under anaerobic conditions will have to clarify whether ROXY9 requires these amino acids for catalysis or for iron-sulfur cluster binding. In an initial experiment to address the repression mechanism of TGA1 by ROXY9 in vivo, a TGA1 version with mutated cysteine residues was constructed and expressed in the tga1 tga4 mutant. This mutant protein is resistant to oxidation and was as active in vivo as wildtype TGA1 regarding hyponastic growth and flowering. However, the experimental setup might not allow the detection of a weak contribution of the TGA1 redox state to hyponastic growth and flowering; thus, this experiment does currently not allow to conclude whether TGA1 is redox-controlled. To test this hypothesis in the future, ROXY9 will have to be overexpressed in tga1 tga4 mutants complemented with wildtype TGA1 and the TGA1 cysteine mutant to find out, whether ROXY9 can still repress the TGA1 cysteine mutant. 6 Contents _____________________________________________________________________ Introduction ........................................................................................... 9 Redox signaling via thiol groups ................................................... 10 Thioredoxins and glutaredoxins .................................................... 13 TGA transcription factors ............................................................. 38 Aim of this thesis ............................................................................ 47 Methods ................................................................................................ 49 Work with organisms...................................................................... 50 Work with DNA ............................................................................. 61 Work with RNA .............................................................................. 69 Work with proteins – basic methods ............................................ 73 Production and analysis of recombinant proteins ...................... 83 Characterization of glutaredoxins .............................................. 101 Results ................................................................................................ 121 Amino acids required for ROXY9 in vivo activity .................... 122 In vitro characterization of ROXY9 ............................................ 131 Towards a mechanism of regulation of TGA1 by ROXY9 ... 164 Discussion ......................................................................................... 173 The CC-type glutaredoxin ROXY9 as an iron-sulfur cluster binding oxidase ............................................................................. 174 Potential regulatory mechanisms of TGA1 by ROXY9 ......... 187 7 Supplementary figures ...................................................................... 191 Supplementary tables ........................................................................ 215 Material ............................................................................................... 225 Organisms ...................................................................................... 226 Oligo nucleotides .......................................................................... 228 Plasmids ......................................................................................... 233 Chemicals ....................................................................................... 247 Commercially available reagents ................................................. 254 Proteins .......................................................................................... 255 Kits ................................................................................................. 257 Lab material ................................................................................... 258 Technical devices .......................................................................... 263 Abbreviations ..................................................................................... 271 Abbreviations ................................................................................ 272 Units and natural constants ........................................................