Funktionale Analyse des CC-Typ Glutaredoxin ROXY19 in Arabidopsis thaliana
Functional Analysis of CC-type glutaredoxin ROXY19 in Arabidopsis thaliana
von Jan Oberdiek
Datum der mündl. Prüfung:2018-05-31
Erschienen:2019-02-05
Betreuer:Prof. Dr. Christiane Gatz
Gutachter:Prof. Dr. Jörg Stülke
Gutachter:Dr. Corinna Thurow
Dateien
Name:Dissertation Jan Oberdiek - Funktionale Anal...pdf
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Zusammenfassung
Englisch
Glutaredoxins are small widespread enzymes that are characterized by the so-called thioredoxin fold. They have an active center which can be reduced by glutathione. The glutaredoxins are divided into three classes according to their active center: class I CPYC-type, class II CGFS-type and class III CC-type (called ROXYs in Arabidopsis thaliana). For class I and II the biochemical properties as oxidoreductases and scaffold proteins for iron-sulfur clusters are described. They contribute to the redox homeostasis of the cell, the regulation of signalling processes, and DNA synthesis. The CC-type class is specific to higher land plants. It has been found to be involved in developmental processes and pathogen defense responses. ROXYs interact physically and genetically with TGA transcription factors. Ectopically expressed ROXY19 suppressed class II TGA-mediated (TGA2, TGA5 and TGA6) expression of ethylene/jasmonic acid-induced defense genes and detoxification genes. The aim of this work was to investigate a possible mechanism for this repression. Possibilities are an enzymatic activity of ROXY19, which allows a redox modification of cysteine residues, or the incorporation of an iron-sulfur cluster as a possible regulatory mechanism. To investigate these glutaredoxin-specific functions, recombinantly expressed ROXY19 from Escherichia coli was used. The affinity to glutathione was detected for ROXY19 with the help of MicroScale Thermophoresis. The affinity was dependent on the CCMC motif and similar to a CPYC type glutaredoxin (GRX370) which had enzymatic activity in the 2-hydroxyethyl disulfide assay. However, ROXY19 had no activity in the 2-hydroxyethyl disulfide assay. A mutation of the active site to the CPYC motif was not sufficient to ensure activity in the 2-hydroxyethyl disulfide assay. The iron-sulfur cluster analysis showed that ROXY19 was able to incorporate an iron species of an unknown type. Further analysis showed that recombinantly expressed ROXY19 from Escherichia coli formed higher molecular oligomers. Reconstitution experiments to determine the type of iron-sulfur cluster with the oligomer and monomer were unsuccessful. Based on Electrophoretic Mobility Shift Assay analysis, a possible mechanism of ROXY19 to control TGA2 activity was discovered. ROXY19 showed a positive influence on TGA2 binding on a radioactive labeled activating sequence-1 promoter fragment (including the TGA binding sequence 5'-TGACG'-3). This feature was independent of the active site and was also observed for the CPYC type glutaredoxin GRX370. ROXY19 induced an additional change in the mobility of the TGA2-activating sequence-1 complex in gel electrophoresis. These data support a ROXY19-TGA2 complex bound to the DNA. The complex might be a possibility to recruit TOPLESS to promoter regions of class II TGA-controlled genes. Huang et al. (2016) observed that plants ectopically expressing ROXY19 in Col-0 background show reduced growth on the xenobiotic 2,3,5-triiodobenzoic acid compared to Col-0 plants. This was based on the ROXY19-mediated repression of detoxification genes. The repression is dependent on the CCMC motif of ROXY19, a mutation to SSMS showed wild-type-like growth. In this work it was shown in planta that the 2,3,5-triiodobenzoic acid susceptible phenotype of plants expressing ROXY19 (Huang et al., 2016) was depending on the first but not the second cysteine of the CCMC motif. Furthermore it was shown, that iron availability had no influence on the ROXY19-mediated repression of class II TGA transcription factor target genes.
Keywords: Arabidopsis thaliana; ROXY19; Glutaredoxin; Redox biology plants