Analysis of the transcriptional repressor function of Arabidopsis glutaredoxin ROXY19

Glutaredoxins (GRXs) are small ubiquitous proteins that are characterized by a thioredoxin (TRX) fold and a glutathione (GSH) reducible active site, which is a CPYC motif in class I GRXs and a CGFS motif in class II GRXs. Biochemically, GRXs can function as thiol-reductases or as scaffold proteins to coordinate Fe-S clusters. Functionally, they are involved in maintaining the reduced state of proteins in the cell and to regulate signaling processes. Only plants encode a third class of GRXs (called ROXYs) which is characterized by a CCMC/S motif. Loss- and gain-of-function experiments have so far revealed that ROXYs regulate both developmental and stress-responsive processes. ROXYs physically and genetically interact with bZIP transcription factors of the TGA family. It has been a long-held hypothesis that ROXYs modulate the activities of corresponding members of the TGA family through redox modification of their cysteine residues. Ectopically expressed ROXY19 suppresses ethylene/jasmonic acid (ET/JA)-induced defense genes through an unknown mechanism that requires the class II TGA transcription factors (namely TGA2, TGA5 and TGA6). The aim of this study was to investigate whether the transcriptional repressor function of ROXY19 involves redox modifications of TGA transcription factors or other targets and to investigate whether its function as a transcriptional repressor can be confirmed by loss of function evidence. Using the protoplast transient expression assays, we identified that ROXY19 represses expression from its own promoter. The capacity of ROXY19 to repress its own promoter in transiently transformed Arabidopsis protoplasts requires TGA-binding sites in the promoter, TGA factors, the C-terminal ALWL motif and a conserved glycine that is required for glutathione binding. Surprisingly, the conserved active site was not important. Moreover, the single conserved cysteine of class II TGA transcription factors is not important for these proteins to confer activation and ROXY19-repressibility to the promoter. Preliminary data obtained from transient expression assays imply that ROXY19, which interacts with the transcriptional co-repressor TOPLESS (TPL) through the ALWL motif, recruits TPL to repress target gene expression. For reasons yet unknown, the active site is required for the negative effects on endogenous ROXY19 and other target genes when ROXY19 is ectopically expressed in transgenic plants. Loss of function evidence of the ROXY function might be hampered by potential redundant function of the 21 members in Arabidopsis. Since only ROXY19 is induced by JA and since it can represses the JA-induced TGA-dependent CYP81D11 promoter when ectopically expressed, we hypothesized that CYP81D11 transcription should be hyper-induced in the roxy19 mutant. However, CYP81D11 transcript levels were not influenced by JA-induced ROXY19. In order to identify potential target genes of ROXY19, the transcriptomes of wild-type, roxy19 and plants ectopically expressing ROXYs were performed. While these experiments did not unravel any genes that were affected by the roxy19 allele, genes from all three phases of the detoxification system were found to be down-regulated in plants ectopically expressing ROXY19. This result is consistent with the well-known function of class II TGA factors as activators of the detoxification pathway upon chemical stress. A motif based analysis revealed that the TGA-binding sites are the over-represented motifs in the promoters of ROXY19-repressed genes. Decreased expression of detoxification genes leads to higher sensitivity of the tga256 triple mutant and plants ectopically expressing ROXY19 towards the xenobiotic chemical TIBA (2,3,5-Triiodobenzoic). However, loss of function analysis showed that plants with mutations in roxy19 and roxy18 (ROXY18 is a closest homolog of ROXY19) do not gain enhanced tolerance to TIBA stress.